Enzymatic Synthesis of Functional Polyesters and Their ......poly(ethylene terephthalate), were...

i

Enzymatic Synthesis of Functional Polyesters and Their

Modification by Grafting Reactions

Dissertation

Zur Erlangung des akademischen Grades

Doctor rerum naturalium (Dr. rer. nat.)

Vorgelegt der

Naturwissenschaftlichen Fakultät II-Chemie, Physik und Mathematik

der Martin-Luther-Universität Halle-Wittenberg

von

Herrn Dipl.-Chem. Toufik Naolou

geb. am 01. August 1979 in Aleppo, Syrien

Gutachter

1. Prof.Dr. Jörg Kreßler

2. Prof. Dr. Carmen Scholz

Halle (Saale), den 23.04.2014

ii

DEDICATION

"To all who sacrificed for dignity, justice and equality in the Arab

world"

Table of Content

iii

Table of Content

Enzymatic Synthesis of Functional Polyesters and Their Modification by Grafting

Reactions __________________________________________________________________ i

Table of Content ____________________________________________________________ iii

Abbreviations __________________________________________________________ vii

Symbols ___________________________________________________________________ ix

Chapter 1- General Introduction ____________________________________________ 1

1.1 Historical perspective ___________________________________________________ 1

1.2 Synthesis and applications of graft copolymers ______________________________ 2

1.2.1 "Grafting-through" strategy_________________________________________________ 2

1.2.1.1 Polycondensation reaction _______________________________________________ 3

1.2.1.2 Homopolymerization of monomers or macromonomers ________________________ 4

1.2.1.3 Copolymerization of monomers and macromonomers _________________________ 4

1.2.2 "Grafting onto" strategy ___________________________________________________ 4

1.2.3 "Grafting from" strategy ___________________________________________________ 5

1.3 Biodegradable polymers and functional polyesters ___________________________ 5

1.4 Enzymatic polymerization. _______________________________________________ 7

1.4.1 Lipase-catalyzed ring opening polymerization __________________________________ 9

1.4.2 Lipase-catalyzed polycondensation ___________________________________________ 9

1.5 "Click" chemistry ______________________________________________________ 11

1.6 Motivation and objective of this work _____________________________________ 15

Chapter 2- Synthesis of Well-Defined Graft Copolymers by Combination of Enzymatic

Polycondensation and “Click” Chemistry ________________________________________ 16

2.1 Introduction _________________________________________________________ 16

2.2 Experimental section __________________________________________________ 17

2.2.1 Materials ______________________________________________________________ 17

2.2.2 Measurements __________________________________________________________ 17

2.2.3 Preparation of 2-(azidomethyl)-2-methylpropane-1,3-diol (AMD) __________________ 18

2.2.4 Typical enzymatic polycondensation procedure ________________________________ 19

Table of Content

iv

2.2.5 Typical procedure for synthesis of poly(2-(azidomethyl)-2-methylpropane adipate)-g-

poly(ethylene oxide) (PAA-g-PEO) __________________________________________________ 19

2.2.6 Synthesis of PAA-g-PEO using enzymatic polymerization and “Click” chemistry in one-pot

process ______________________________________________________________________ 21

2.3 Results and discussion _________________________________________________ 21

2.3.1 Enzymatic prepapration of poly(2-(azidomethyl)-2-methylpropane adipate) (PAA). ____ 21

2.3.2 Synthesis of PAA-g-PEO using “Click” chemistry ________________________________ 24

2.3.3 Synthesis of PAA-g-PEO in sequential one-pot reaction (PAA-g-PEOop) ______________ 26

2.3.4 Surface tension measurements _____________________________________________ 27

2.3.5 1H NMR spectroscopy in water and in THF ____________________________________ 28

2.3.6 Dynamic light scattering __________________________________________________ 29

2.3.7 Langmuir trough measurements ____________________________________________ 30

2.4 Conclusions __________________________________________________________ 32

Chapter 3- Utilization of Poly(glycerol adipate) to Synthesize Graft Copolymers and

Polymeric Analogues of Glycerides ____________________________________________ 33

3.1 Introduction _________________________________________________________ 33


3.2.1 Materials ______________________________________________________________ 34

3.2.2 Synthesis of poly(glycerol adipate) (PGA) _____________________________________ 35

3.2.3 Acylation of PGA backbone with fatty acid chains ______________________________ 36

3.2.4 Synthesis of alkyne modified poly(glycerol adipate) (PGA-Alkyne) __________________ 36

3.2.5 Synthesis of Poly(glycerol adipate)-g-Poly(ethylene oxide) PGA-g-PEO ______________ 37

3.2.6 Polymer nanoparticle preparation __________________________________________ 37

3.2.7 Differential Scanning Calorimetry ___________________________________________ 37

3.2.8 Transmission electron microscopy (TEM) _____________________________________ 38


3.3.1 Synthesis of poly(glycerol adipate) (PGA) backbone _____________________________ 38

3.3.2 Temperature dependence of rigioselectivity ___________________________________ 41

3.3.3 Synthesis poly(glycerol adipate)-g-poly(ethylene oxide) (PGA-g-PEO) _______________ 42

3.3.4 Modification of PGA backbone with fatty acids ________________________________ 44

3.3.5 DSC measurements ______________________________________________________ 45

3.3.6 Thermogravimetry _______________________________________________________ 46

3.3.7 Transmission electron microscopy __________________________________________ 46

3.4 Conclusions __________________________________________________________ 47

Table of Content

v

Chapter 4- Synthesis and characterization of graft copolymers able to form

polymersomes and worm-like aggregates ______________________________________ 49

4.1 Introduction _________________________________________________________ 49


4.2.1 Materials ______________________________________________________________ 50

4.2.2 Synthesis of poly(glycerol adipate)-g-poly(ε-caprolactone) (PGA-g-PCL) _____________ 51

4.2.3 Synthesis of alkyne-modified poly(glycerol adipate)-g-poly(ε-caprolactone), (PGA-g-(PCL-

alkyne)) ______________________________________________________________________ 51

4.2.4 Synthesis of PGA-g-(PCL-b-PEO) using click chemistry ___________________________ 53

4.2.5 Synthesis of -hydroxy--alkyne end functionaleized poly(ε-caprolactone) (Alkyne-PCL) 53

4.2.6 Synthesis of poly(ε–caprolactone)-b-poly(ethylene oxide) (PCL-b-PEO) ______________ 53

4.2.7 Procedures _____________________________________________________________ 54

4.2.8 Micelle preparation ______________________________________________________ 55

4.2.9 Worm-like aggregates ____________________________________________________ 55

4.2.10 Fluorescence microscopy (FM) of worm-like aggregates _________________________ 55

4.2.11 Transmission electron microscopy (TEM), and scanning electron microscopy (SEM) ____ 56


4.3.1 Synthesis and characterization of PGA-g-(PCL-b-PEO) and PCL-b-PEO _______________ 56

4.3.2 Dynamic light scattering (DLS) ______________________________________________ 61

4.3.3 Micelle characterization by 1H NMR spectroscopy ______________________________ 62

4.3.4 Surface tension measurements _____________________________________________ 65

4.3.5 Scanning electron microscopy (SEM)_________________________________________ 65

4.3.6 Preparation and characterization of worm-like aggregates _______________________ 66

4.4 Conclusions __________________________________________________________ 70

Chapter 5- The Behavior of Poly(ɛ-caprolactone) and Poly(ethylene oxide)-b-Poly(ɛ-

caprolactone) Grafted to a Poly(glycerol adipate) Backbone at the Air/Water Interface _ 72

5.1 Introduction _________________________________________________________ 72


5.2.1 Materials ______________________________________________________________ 73

5.2.2 Surface pressure measurements ____________________________________________ 73

5.2.3 Brewster angle microscopy (BAM) __________________________________________ 74

5.2.4 Deposition of Langmuir–Blodgett (LB) films ___________________________________ 75


5.3.1 The behavior of linear and grafted PCL at the A/W interface ______________________ 75

5.3.2 The behavior of linear and grafted PCL-b-PEO at the A/W interface ________________ 82

Table of Content

vi

5.3.3 Langmuir Blodgett films ___________________________________________________ 85

5.4 Conclusions __________________________________________________________ 87

Chapter 6- Summary ____________________________________________________ 89

References ____________________________________________________________ 92

Acknowledgments _____________________________________________________ 105

Curriculum Vitae ______________________________________________________ 106

Abbreviations and Symbols

vii

Abbreviations

AFM Atom Force Microscopy

ATRP Atom Transfer Radical Polymerization

Asp Aspartic Acid

CAC Critical Aggregation Concentration

CuBr Copper bromide

CMC Critical Micellization Concentration

CAL-B Lipase B derived from Candida Antarctica

CRP Controlled Radical Polymerization

CuAAC Copper (I) Catalyzed Huisgen 1,3-Dipolar Azide-

Alkyne Cycoloaddition, "Click" Chemistry

DCC dicyclohexylcarbodiimide

DCM Dichloromethane

DLS Dynamic Light Scattering

DMA Dimethy Adipate

DMAP 4-(Dimethylamino)pyridine

DMF Dimethylformamide

DMSO-d6 Deutrated dimethylsulfoxide

DP Degree of Polymerization

DSC Differential Scanning Calorimetry

DVA Divinyl Adipate

FM Fluorescence Microscopy

His Histidine

HDA Hetro-Diels-Alder

Et3N Triethylamine

EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

FDA Food and Drug Administration

FTIR Fourier Transform Infrared

NMR Nuclear Magnetic Resonance

mPEO-N3 Azide-Terminal Poly(ethylene oxide)-

Monomethylether

mmA Mean Molecular Area

MWCO Molecular Weight Cut Off

PAA Poly(2-(azidomethyl)-2-methylpropane adipate)


viii

PAA-g-PEO poly(2-(azidomethyl)-2-methylpropane adipate)-g-

poly(ethylene oxide)

PAA-g-PEOop poly(2-(azidomethyl)-2-methylpropane adipate)-g-

poly(ethylene oxide) in "one pot"

PCL Poly(ε-caprolactone)

PDI Polydispersity

PEO Poly(ethylene oxide)

PGA Poly(glycerol adipate)

PMDETA N,N,N′,N′′,N′′-pentamethyldiethylenetriamine

RAFT Reversible Addition Fragmentation Chain Transfer

ROP Ring Opening Polymerization

SEC Size Exclusion Chromatography

SEM Scanning Electron Microscopy

TEM Transmission Electron Microscopy

TGA Thermogravimetric Analysis

THF Tetrahydrofuran


ix

Symbols

π Surface Pressure

δ Chemical Shift

Scattering Angle

λ Wavelength

Γ Decay Rate

Specific Enthalpy of Melting

Dapp Apparent Diffusion Coefficient

q Scattering Vector

n0 Refractive Index

Mn Number Average Molar Mass

Mw Weight Average Molar Mass

TC Crystallization Temperature

Tm Meting Temperature

γ Surface Tension

T Temperature

H0

mic Standard Enthalpy of Micellization,

XDSC Degree of Crystallinity

εs Static Elasticity

Chapter 1 General Introduction

1

Chapter 1

General Introduction

1.1 Historical perspective

It is surprising that industry began to produce polymers before scientists even knew the

fundamental understanding of structure and nature of polymers. During that time development

of polymer materials was carried out by "trial and error" which might be described also as

"Edisonian" fashion. Polymer products appeared during that time either by modification of

natural polymers, such as modification of cellulose to produce celluloid, or via syntheses of

new polymers such as Bakelite which was produced by the reaction of phenol and

formaldehyde.1 The concept of high molecular compounds "macromolecular chemistry" was

first introduced by the German organic chemist Hermann Staudinger in 1917. The concept

was further expanded to become "polymer science" by Hermman F. Mark in order to cover

the organic chemistry and physical chemistry of polymers.2 In the thirties of the last century,

many synthetic polymer products, such as polystyrene, poly(methyl methacrylate),

polyethylene, poly(vinyl chloride), polybutadiene, polychloroprene, nylon-6,6, and

poly(ethylene terephthalate), were developed as a result of the urgent need to find alternatives

for natur materials which were of short supply. Silicon rubber was produced during the forties

of the last century, whereas polyolefins beside polycarbonate production started during 1950s.

The year 1953 saw the development and production of polyethylene under low pressure using

Ziegler catalyst whereas in 1954 Giulio Natta produced for the first time high molar mass

polypropylene.3 The anionic polymerization technique was also invented by Szwarc et al. in

1956.4 This synthetic approach especially enabled polymer chemists for the first time to

control for number average molar mass (Mn), the degree of polymerization (DP), and the

polydispersity index (PDI). Additionally, this technique opened the door to synthesize block

copolymers since macro-anions formed during the polymerization exhibit living properties.

Living cationic polymerization was developed in the 1970s and 1980s.5–7

The first report

about enzymatic polycondensation was demonstrated in the middle of the 1980s. This

technique was further applied to polymerize cyclic ester monomers (lactones) in 1993.8

Enzymatic polymerization indeed enabled polymer chemists to prepare functional polyesters

under mild conditions without the need of the protection/deprotection steps. The early 1990s

saw the introduction and development of novel approaches to synthesize polymers called


2

controlled radical polymerization (CRP) was possible.9 This approach covers three novel

polymerization techniques, including reversible addition-fragmentation chain transfer

polymerization (RAFT),10

atom transfer radical polymerization (ATRP),11,12

and nitroxide-

mediated radical polymerization (NMP).13

In conclusion, substantial developments were indeed made in different fields of polymer

science during the last century such as, finding new techniques to synthesize polymers with

various architectures, understanding the mechanisms, kinetics of different polymerization

techniques and or the physical properties of polymers with various architectures under

different conditions. In fact, our nowadays knowledge in organic synthesis and polymer

chemistry enable scientists to prepare virtually any monomer and its associated polymer.14

According to the new synthesis techniques which have appeared during the past twenty years,

many polymers with complex architectures have been synthesized such as, cyclic, multicyclic,

dendritic, hyperbranched, star, graft, and arborescent.15

It should be noted that synthesis of

such complex topologies before that time would not have been possible.

1.2 Synthesis and applications of graft copolymers

Graft polymers or copolymers belong to the family of nonlinear, branched segmented

copolymers in which the polymer backbone has a number of side chains of different chemical

nature.16

Graft polymers have attracted increasing attention due to their unusual properties

caused by confined and compact structures in comparison with the identical linear counterpart

having similar molar mass.17

Such unique structural characteristics make graft copolymers

candidates for a lot of advanced applications, such as preparation nanostructures,18

preparation of hybrid nanostructure,19

biomedicine, 20

super soft elastomers,21

and in

photonics.22

The development of advanced synthetic techniques such as living/controlled

polymerization, and "click" chemistry facilitate the synthesis of graft copolymers with well-

defined structure and low molecular polydispersity. Graft copolymers can be synthesized via

three main synthetic routes as shown in Figure 1.1.

1.2.1 "Grafting-through" strategy

This method is based on polymerization of macromonomers having a α-polymerizable

group which offers the ability of controlling the grafting density by controlling the ratio

between monomers and macromonomers during the polymerization process. Additionally, it

is possible using this technique to synthesize graft copolymers having well-defined chemical

structures since the characterization of the side chains occurs prior to the grafting reaction.


3

Figure 1.1 The main synthetic route to prepare graft copolymers.

Using the grafting-through method can also produce graft copolymers having 100% grafting

density (every repeating unit has one side chain). Unfortunately, grafting reaction does not

usually include all of the macromonomers exists in the reaction vessel. Even by using

effective polymerization techniques such as ring-opening metathesis polymerization (ROMP),

the complete conversion of macromonomers seems to be also not feasible, mostly due to the

low reactivity of macromonomers and large hindrance between α-functionalized

macromonomers and the reactive sites in the propagating graft copolymer. Thus, it is always

necessary to purify the final product from unreacted macromonomers which is certainly time

consuming.

Preparation of graft copolymers by "grafting through" strategy are carried out using three

synthetic pathways 23

1.2.1.1 Polycondensation reaction

Many scientists have reported the direct synthesis of comb-like polyesters having aliphatic

alkyl side chains by utilizing monomers that can participate in condensation reactions and

having n-alkyl chains within its structure. Lenz et al. reported the synthesis of poly(2-n-alkyl-

1,4-phenylene terephthalate) through polycondensation processes between terephthalic acid


4

and 2-n-alkyl-hydroquinone.24

Additionally, Watanabe et al. reported on the preparation of a

series of rigid-rod polyesters having aliphatic side chains.25

1.2.1.2 Homopolymerization of monomers or macromonomers

Rehberg and Fisher have introduced the first comb-like polymer in 1944 based on poly(n-

alkyl acrylate)s which was prepared radically.26

Thereafter, many comb-like polymers were

reported such as poly(α-olefine)s, poly(n-alkyl methacrylate)s, poly(n-alkyl acrylamide)s,27

and poly(n-alkyl itaconate). However, controlling the molar mass and regularity of molecular

chains always presents a difficult task when scientists utilize this conventional polymerization

technique to prepare comb-like polymers.

1.2.1.3 Copolymerization of monomers and macromonomers

A series of graft copolymers having poly(ethylene glycol) (PEG) as a side chains have been

synthesized by "grafting through" strategy by polymerize reactive monomers and ω-

functional PEG, such as, poly(styrene)-g-PEG,28

poly(methyl methacrylate)-g-PEG,29

poly(butyl methacrylate)-g- PEG.30

1.2.2 "Grafting onto" strategy

Grafting-onto strategy is based on grafting the end-functional polymer onto a linear backbone

through the reactive sites present on each monomer units.23

Utilization of the "grafting-onto"

route yields well-defined graft copolymers since precise characterization of side chains and

the backbone can be carried out prior to the grafting reaction. However, this technique has

also several drawbacks, including limited grafting density, and the necessity of purifying

uncoupled side chains from the grafted copolymers. The grafting density varies in the case of

"grafting-onto" technique according to the chemical structure of the utilized side chains where

for the more bulkier side chains such as poly(styrene), poly(butyl acrylate), and poly-(n-butyl

acrylate)-b-poly(styrene) grafting densities are smaller than 50%.31

Such a result is the

consequents to the steric hindrance caused by the attached side chains to the reactive side on

the polymer backbone. In contrast, using relatively thinner side chains such as poly(ethylene

oxide) (PEO) will result in brush polymers with grafting density up to 88%.31

The main key

for the successful preparation of graft copolymers via "grafting-onto" strategy is to use

efficient coupling reactions between the end-functionalized macromonomer and the functional

backbone. In general, graft copolymers using "grafting-onto" approach can be prepared using

two methods:23

i) covalent "grafting-onto" via chemical coupling reaction between the side

chains and polymer backbone,32

or ii) non-covalent "grafting-onto" by supramolecular


5

chemistry assembly processes through metal-ligand coordination,33

π-π interaction, hydrogen

bonding,34

and or ionic interaction.35

1.2.3 "Grafting from" strategy

This method can be defined as the process of initiating the polymerization of side chains from

the predetermined initiation sites on the polymer backbone (macroinitiator), which is either

existing within the structure of monomers before polymerization or being introduced onto the

backbone afterwards.23

Many reports have appeared describing this technique to synthesize

well-defined graft copolymer. 36,37 Graft copolymers prepared by this method are characterized

by high grafting density and a narrow molar mass distribution. Using controlled radical

polymerization such as ATRP or ROMP for grafting reaction, yield well-defined graft

polymers since the low concentration of instantaneous propagating species decrease

significantly the coupling and termination reactions. Furthermore, the gradual growth of the

side chains could also decrease notably the steric effect which normally exists in the case of

"grafting-onto" and grafting-through" routes.17

Additionally, it is not necessary to use dialysis

and fractionation methods to purify the resulting graft copolymers since there is no unreacted

macroinitiator. Nevertheless, this technique also suffers from many drawbacks such as

carrying out grafting reactions in highly diluted systems and/or the relatively low monomer

conversion which is necessary to avoid crosslinking reactions between the macroinitiators. In

fact, working under such conditions leads to a significant waste of monomers, solvent and

also long reaction time.

It is worth mentioning here that the synthesis of amphiphilic graft copolymers having two

different side chains have been previously reported , which can be obtained either by using

only one type of grafting strategy38

or by a combination of two types,39

e.g. the combination

of “grafting from” and “grafting onto”.40

1.3 Biodegradable polymers and functional polyesters

Over the past century scientist have focused on inventing new materials to meet the

needs of modern life and further to find alternative methods to decrease production costs.

However, many environmental problem emerge as a result of using toxic catalysts during

polymer production or due to difficulties of disposal plastic wastes.41

Thus, researchers have

developed over the last three decades eco-friendly reactions and materials in order to achieve

sustainability. Biodegradable polymers came to the market as alternative products to

nondegradable conventional polymers for packaging and biomedical applications.42

Aliphatic


6

polyesters are considered to be the most of used biodegradable polymers especially for

biomedical applications.43

However, mechanical, biological, and physical properties of these

polyesters are not always meeting the technical needs of many applications.44

The presence of

pendent functional groups on the polyesters backbone, however, can have a significant impact

on properties for potential applications.45–47

For instance, polyesters with pendant cationic

groups could be used to make surfaces antibacterial and for gene therapy.48,49

Furthermore, it

has been demonstrated that the covalent conjugation of drug to the functional groups of

amphiphilic block copolymers, which are designed to be used as nano-size drug carriers, is

much better than the classic physical encapsulation as it prevents the leakage of drugs by

diffusion from micelles.50

Additionally, introducing functional groups onto the blocks that

forms the core of micelle can enable adjusting the encapsulation and release properties of

micelles or nanoparticles by some special interactions between the block forming the core and

the drug, such as hydrogen bond,51

π-π interaction,52

electrostatic complexation,53

and some

chemical reaction.54

Moreover, introducing pendent functional groups to the polyester can

enhance its degradation properties,55

offering opportunities to attach the polymer backbone

with biological active components or increase the cell/matrix interactions in the tissue

engineering field.56

Despite these advantages, introduction of functional groups to the

polyester backbone is still a challenge.57

In general, functional polyesters can be synthesized

via

i) anionic activation of linear polyesters using non-nucleophilic bases such as lithium

diisopropyl amide to form polycarbanion on which electrophiles can be easily

attached. 58

ii) ring-opening polymerization of functional lactones and lactides.59–62

iii) ring opening polymerization (ROP) of functional O-carboxyanhydrides.63

iv) catalyzed polycondensation of polyfunctional monomers.64,65

In terms of limitations, using the first strategy (i) to synthesize functional polyesters could

cause simultaneous main-chain degradation.66

On the other hand, using ring opening

polymerization of lactones to synthesize functional polyesters offers a lot of attractive

advantages,67

such as the reaction proceeds in one direction without generating leaving groups

during the course of reaction,68

high molar mass polyesters within short time, low

polydispersity and it proceeds by chain growth mechanism which can control its telechelic

functional groups.69

Ring opining polymerization of O-carboxyanhydrides has the same

advantages as lactones except that carbon dioxide is released during the polymerization

reaction, readily to evaporate at reaction temperatures. However, it is not possible using ring


7

opening polymerization to synthesize polyesters with free hydroxyl, carboxylic acid or

mercapto pendent functional groups in one step since these functional groups are considered

as initiators for ring opining polymerization. Actually, polyesters with free hydroxyl,

carboxylic acid or mercapto pendent functional groups can be synthesized via ring opining

polymerization either by polymerization of protected cyclic monomers (lactones, lactides and

O-carboxyanhydrides) followed by deprotection reaction, 55,63,70–74

or by synthesizing

polyesters which contain ketone groups within the polyester backbone followed by

hydrogenation reaction of the ketone bond to form hydroxyl groups.57,75,76

The protection

steps of monomers prior to polymerization, in addition to possible degradation of the

polyester backbone during the deprotection step are considered the major disadvantages of

this synthetic route. In contrast, functional polyesters can be synthesized in one step by

enzymatic polycondensation reactions without the need of protecting monomers.

1.4 Enzymatic polymerization

Enzymatic polymerization is defined as " the in vitro polymerization of artificial substrate

monomers catalyzed by an isolated enzyme via nonbiosynthetic (nonmetabolic) pathways".77

In vitro enzymatic catalysis was first reported by a Polish chemist in the 1930s for the

synthesis of esters in organic media.78

However, these results did not attract much of the

attention of scientists, until Klibanov et al. reported similar reactions in 1984.79

Since then,

more interest has been given for this novel technique in the field of organic synthesis due to

its ability to control the rigioselectivity and stereochemistry of the products, major limitation

in organic synthesis techniques.77

The enzymatic polymerization, however, was introduced in

the late 1980s and extensively investigated in the following two decades. Lipase-catalyzed

polyester synthesis is considered to be one of the most extensively investigated topics in

enzymatic polymerization. Enzymatic polycondensation route, especially lipase/esterase-

catalyzed polymerization, has many advantages compared to conventional chemical routes, 80–

82 such as

i) mild reaction conditions which can reduce the energy consumption of the overall

process and the possibility of polymer chain degradation that might occur by working at

high temperatures.

ii) It does not require protection-deprotection steps due to the high ability of enzymes to

control chemo-, enantio-, and regioselectivity of the products.

iii) high catalytic activity.

iv) fewer byproducts (considered as clean process).

v) the enzyme is recyclable when it is immobilized, which reduces the catalyst costs.


8

vi) sustainability of the enzyme.

vii) enzymes are derived from renewable resources.

In principle, lipases are responsible to catalyze the hydrolytic cleavage of fatty acid triacyl

glycerol ester in vivo. However, lipases can in vitro form ester bond instead of breaking it

when the reaction is carried out in anhydrous media and the resulting by-products are

removed. Lipases are considered to be the most popular biocatalysts for enzymatic

reactions.83

In particular, lipase derived from Candida Antarctica lipase B (CAL-B) is

considered to be one of the most commonly used enzyme in the field of enzyme-catalyzed

condensation polymerization for polyesters.77,80

This enzyme is commercial available under

the trade name Novozyme 435, which consists of physically adsorbed CAL-B within the

macropores of poly(methyl methacrylate-co-butyl methacrylate) resin. In general, the

immobilized enzymes can facilitate their removal from the final product, allowing for its

reuse. Additionally, immobilization of enzymes can also improve their properties like

stability, activity, their selectivity to non-natural substrates, and enantioselectivity.84

Figure 1.2 Main synthetic routes to prepare polyesters using enzyme as a catalysts. 77

There are two main routes for enzymatic synthesis of polyesters which are shown in Figure

1.2.77


9

1.4.1 Lipase-catalyzed ring opening polymerization

This synthetic technique was first introduced in 1993 by Kobayashi et al. by carrying out ring

opening polymerization of ε-caprolactone and -valerolactone.85

Utilizing this route to

produce polyesters has been extensively investigated and high molar mass polyesters were

produced under relatively mild conditions. Additionally, this polyaddition reaction is

performed with no or very limited amounts of side-reactions which makes it possible to

control the polymer properties such as molar mass, molar mass distribution, and polymer end

groups.43

Interestingly, it has been illustrated that reaction kinetics and the achievable molar

mass in lipase–catalyzed ROP increase by increasing the ring size of lactones in contrast to

traditional chemical methods which has led to the polymerization of macrolactones that are

derived from natural sources.86

For instance, poly(pentadecalactone) was enzymatically

synthesized using ω-pentadecanolide as a monomer with a molar mass of up to 150 000 g

mol-1

. Controlling the end-group of a polymer is considered as a critical issue in polymer

chemistry especially in the case of synthesizing amphiphilic polymers. Thus, this topic has

been extensively investigated in the lipase-catalyzed ROP of lactones. Using functional

alcohol as initiator was applied in this technique to incorporate functional groups into the

polymer chain. However, a mixture of cyclic species, water initiated polymer chains and

polymer chains with desired functional groups were obtained. Actually, the maximum degree

of obtained functionality was 95% even when stringent drying conditions were applied.87

1.4.2 Lipase-catalyzed polycondensation

Enzymatic polycondensation is defined as "enzyme catalyzed esterification and

transesterification between diacids or their activated esters with diols or self-polycondensation

of hydroxyacids in non-aqueous media". Like any condensation reaction, enzymatic

polycondensation is usually a reversible reaction since it is accompanied by low molar mass

compounds as byproduct. Removal of this byproduct is considered as a critical factor to shift

the equilibrium towards the products. Monomers having activated acyl donors, such as oxime

ester, thioester, and anhydrides, have been used instead of traditional carboxylic acids in order

to increase the activity of the monomers towards enzymatic polymerization and to facilitate

removing byproducts. Using enol esters , however, such as vinyl esters seems to be the most

efficient synthetic route since they release unstable enols as a byproduct which tautomerizes

readily to give the corresponding aldehydes or ketones.83

CAL-B is composed of 317 amino acid residues and its structure was determined in 1993.88

Its active center has a catalytic triad, serine (Ser105)-histidine (His224)-aspartic acid


10

(Asp187). A large hydrophobic pocket exists above the Ser105-His224-Asp224 triad whereas

a medium-sized pocket exists below it. The accepted mechanism by which CAL-B catalyzes

transesterification reaction to yield polyester is illustrated in Figure 1.3. The catalyst site in

the active center of CAL-B is the –CH2OH of the Ser residue. The imidazole group of the His

residue pulls a proton from –CH2OH which augments the nucleophilicity of the oxygen in

order to attack the carbonyl group in the substrate.

Figure 1.3 Illustration of lipase-catalyzed transesterification mechanism.89

Meanwhile, the carboxylate group of the aspartic acid residue helps the imidazole group to

pull a proton of the Ser residue and trasnlocate it to the substrate and subsequently the

corresponding alcohol will be released from the substrate. As a result, a covalent bond will be

formed between the enzyme and the substrate to get the acyl-enzyme intermediate.

In the deacylation step, the nucleophile (which is generally water, or alcohol, or amine) will

attack the acyl-enzyme carbonyl group. In this process the proton of the nucleophile is


11

transferred to the His residue of the enzyme. This hydrogen is transferred again to the Ser

alkyl oxygen which causes the weakness of the enzyme-product complex bond which

ultimately releases the reaction product from the enzyme allowing for its regeneration.89,90

Nevertheless, using the enzymatic polycondensation route to prepare polyesters also suffers

from several drawbacks including43

i) Like any conventional polycondensation, enzymatic polycondensation requires a

precise stoichiometric balance between the hydroxyl groups and reactive acids with high

conversion ratio in order to obtain high molar masses.

ii) The need to remove resulting by-product(s) in order to shift the reaction equilibrium.

iii) The inability to control of the telechelic functional groups since polycondensation

proceeds via step-growth mechanism.

In contrast to lipase-catalyzed ROP, using the enzymatic polycondensation route has a great

benefit for the preparation of polyesters with free pendant functional groups in one step

without the need of the protection/deprotection steps which might otherwise cause

degradation of the polymer backbone. The chemo-, and regio-selectivity of lipase are

considered to be the main reason behind their use. Accordingly, functional polyesters have

been enzymatically synthesized with free pendent groups, such as alcohol 91,92

, mercapto,93

and carboxylic acid,94

which cannot be synthesized in one step by enzymatic ROP.

Furthermore, this technique allows for the production of polyesters form renewable resources

as e.g. glycrol of sugars.

1.5 "Click" chemistry

In 2001, Sharpless introduced the concept of "click" chemistry and defined it as a "set of

powerful, highly reliable, and selective reactions for rapid synthesis of useful new compounds

and combinatorial libraries".95

Any chemical reaction can be classified as a "click" reaction if

it is characterized by the following features: modular and wide in scope, highly efficient with

high yield, resulting in no or unoffending byproduct, stereospecific, its starting materials and

reagents are readily available, insensitive to the type of solvent, and easily to be purified.96

A

particular interest has been paid during the last decade to the applications of "click" reactions

in polymer synthesis since it can solve many of the critical problems which encounter this

field.97

Accordingly, utilization of "click" reaction in polymer synthesis facilitates the

preparation of many novel polymers with complex chain topologies (ie, graft, star,

dendrimers, and cyclic) or block copolymers which cannot be synthesized by conventional

polymerization pathways.


12

Figure 1.4 The mechanism proposed for CuAAC "click" reaction.98

w here L is ligand, B is a

base.

Copper(I) catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC) was introduced shortly

after 2001.99

This reaction has become within a few years the most important "click" reaction

as it plays a particular important rule in organic and macromolecular synthesis. Azide and

alkyne groups are stable in the presence of electrophiles or nucleophiles and they are almost

completely unreactive towards biological molecules.99

Additionally, both groups are not able

to form a significant hydrogen bonds and they are relatively nonpolar, and thus are unlikely to

change significantly the properties of compounds onto which they are attached.

The mechanism proposed for CuAAC click reaction is shown in Figure 1.4. The mechanism

is explained as a stepwise process which begin with the formation of Cu(I) acetylide species

via the π complex followed by azide complexation and cyclization. Finally, the triazole-

copper derivative becomes protonated and subsequently dissociates to yield the final product

beside the catalyst. Different compounds have been used as a ligand in this reaction to


13

dissolve the catalyst, such as amines, phosphines, triazoles, and pyridine. It has been reported

that the type of ligand has a significant effect on the kinetics of the "click" reaction explained

by decreasing the oxidation of Cu(I), and promoting the formation of the copper-acetylide

complex.100

However, the product which participates in CuAAC reaction is not suitable for

biological applications due to the presence of transition metal traces. Significant interest has

been made in the last couple of years in developing new "click" reactions that do not require

metal catalysts while still exhibiting all "click" chemistry criteria.101

One elegant approach is

the reaction between azide and strained cycloalkynes developed by Bertozzi et al.102

However, the reaction rate is relatively low in comparison with the CuAAC reaction. Thus,

electron withdrawing substituents are added at the α-position of this triple bond (Figure 1.5a).

Cycloaddition reactions of unsaturated species have been proposed also as alternative "click"

reactions to the CuAAC reaction. As an example, Diels-Alder [4+2] cycloaddition between

maleimide and anthracene derivatives has been proposed and used successfully in the polymer

synthesis as metal free "click" reaction (Figure 1.5b).103

The necessity of high temperature

application, however, for the coupling reaction limits the usage of this synthetic pathway to

polymeric structures that are thermally unstable. The groups of Kowollik and Stenzel reported

recently an alternative hetero-Diels-Alder (HDA) route as a coupling reaction between

terminal electron-deficient thiocarbonylthio group of polymers which are produced by RAFT

polymerization technique and an appropriate diene (Figure 1.5c). The dienophilicity of the

dithioester end group is further increased in HDA by utilization of trifluoroacetic acid or zinc

chloride as a catalyst which enhances the electron-withdrawing effect of the Z-group. This

reaction proceeds to a high conversion at reaction temperature of 50 °C and reaction time

between 2 and 24 h.104

An efficient and ultrafast hetero-Diels-Alder reaction has also been

reported by the utilization of more reactive cyclopentadiene compared with linear dienes

(Figure 1.5d).105

Surprisingly, a high coupling yield was obtained within just a few minutes at

ambient temperature without the need of any catalyst addition. Thus, this type of "click"

chemistry is expected to become an important synthetic route in polymer synthesis.


14

Figure 1.5 Examples of click reactions employed commonly in polymer synthesis. (a) strain-

promoted azide-alkyne cycoloaddition. (b) Diels-Alder [4+2] cycloaddition

between maleimide and anthracene derivatives. (c) Hetero-Diels-Alder HDA. (d)

Ultra hetero-Diels-Alder reaction. (e) Thiol-ene click reaction.15

A series of thiol-based reactions with unsaturated bonds have highlighted recently as a

powerful coupling chemistry (Figure 1.5e). Typically, these reactions can be classified into

two main categories depending on the chemical nature of unsaturated bond and reaction

conditions: i) thiol-ene reaction by ionic mechanism (Michael-type addition) or ii) by free

radicals (thermally initiated or by UV-light). Thiol based "click" reactions are considered as

simple, highly reactive, need short reaction times and can be performed at ambient

temprature.106


15

1.6 Motivation and objective of this work

As already mentioned at the beginning of this chapter, there is currently a great interest to

investigate the potential applications of macromolecules which have complex topologies,

particularly graft copolymers. Actually, many reports have recently appeared showing

superior properties of graft copolymers compared to the classical linear block copolymers,

considering their ability to form more stable micelles with low critical micelle concentration

(CMC), high drug loading, low melting point Tm and small crystallization degree.107–110

Such

features suggest the use of graft copolymers in the field of drug delivery systems instead of

the classical block copolymers. To achieve such target the biodegradability and the

biocompatibility of the synthetic graft copolymer should also be considered.

The main objective of this work is to synthesize well-defined graft copolymers that are

suitable for drug delivery applications and to investigate their properties. In order to achieve

this goal, a series of novel graft copolymers is synthesized where their main backbones are

composed of biodegradable aliphatic polyesters. In fact, the presence of free pendent

functional groups on the polyester backbone used in this study is found critical as it facilitates

the grafting process using different coupling reactions. Thus, several approaches are

suggested and applied to obtain functional polyesters with functional groups in each repeat

unit. CAL-B is used as a catalyst for the polyester preparation, found suitable for products that

can be used in the biomedical field. Poly(ethylene oxide) (PEO) chains are mainly attached as

side chains in these reactants since PEO are water soluble polymer that can form a

hydrophilic shield protecting it from the immune system recognition and thus prolonging its

in vivo circulation time.111

Additionally, a comparison is made between the differences in

properties achieved by synthesizing graft copolymers that have hydrophobic/hydrophilic

block copolymers as a side chains versus synthesizing linear block copolymers that have

identical chemical composition as the grafted chains. The comparison reveals the difference

of the polymer properties in water and at the air/water interface. Such a comparison is aimed

to find the potential applications for using graft copolymers instead of conventional linear

block copolymers.

Chapter 2 Synthesis Graft Copolymer in Sequential "One-Pot"

16

Chapter 2

Synthesis of Well-Defined Graft Copolymers by Combination of Enzymatic

Polycondensation and “Click” Chemistry

2.1 Introduction

Linear aliphatic polyesters represent one of the most important groups of biocompatible

and biodegradable polymers having a huge versatility with respect to physical, chemical, and

biological properties.112

The presence of pendant functional groups at the polyester backbone

can be used for further modification of polymer properties. Anionic activation of linear

polyesters using non-nucleophilic bases to form polycarbanion on which electrophiles can

easily be attached,113–115

ring-opening polymerization of substituted lactones,59–61,64,116–121

and

catalyzed polycondensation of polyfunctional monomers65,91,92,122,123

is the main strategy used

to synthesize aliphatic polyesters with pendant functional groups. When carrying out the

polycondensation of glycerol with derivatives of dicarboxylic acids, it is necessary to use a

chemoselective catalyst to obtain linear poyesters.80

Using, for example, lipase from Candida

antarctica type CAL-B as a chemoselective enzyme can result in linear polyesters because the

enzyme favors the condensation process of primary alcohols rather than secondary

alcohols.124

When polyesters with pendant groups are used to synthesize well-defined graft

copolymers, functional groups should meet strict requirements such that

(i) They must be reactive enough to attach other polymer (oligomer) chains quantitatively

under large steric restrictions.

(ii) They should undergo the coupling reaction in one step under mild conditions to avoid

any degradation of the polyester backbone.

(iii) The coupling reactions should be selective only for this functional group, which

means that protection/deprotection steps are not required for other functional groups

present on the polymer backbone.

Recently, the concept of copper-catalyzed azide−alkyne cycloaddition CuAAC, “click”

reaction, which was first introduced by Meldal et al.125

and Sharpless et al.,126

meets exactly

the previously described requirements to synthesize graft copolymers using the “grafting

onto” protocol.31

It seems reasonable to use monomers containing clickable functional groups

for enzymatic polycondensation rather than introducing clickable groups by polymer

analogous reactions, which might also attack the sensitive ester groups of the polymer


17

backbone.127

Therefore, this approach is based on the polycondensation of a N3-containing

diol that is used for polycondensation with divinyl adipate (DVA). This polymer is then used

for “click” reaction with monoalkyne-functional poly(ethylene oxide) (alkyne-PEO, Mn = 750

g/mol). This reaction is based on the Huisgen 1,3-dipolar cycloaddition chemistry.128

According to the properties of PEO such as biocompatibility and water solubility 61

and for

applications of aliphatic polyester in bioresorbable medical applications,129

connecting both

species to graft copolymers should lead to potential biomedical materials. Here the

aggregation behavior of the polymers in water is analyzed by dynamic light scattering (DLS),

and the behavior at the air/water interface is studied by surface tension measurements and by

Langmuir trough experiments.

2.2 Experimental section

2.2.1 Materials

All chemicals were purchased from Sigma-Aldrich unless otherwise stated. Hydrobromic

acid, n-hexane, sodium azide, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF),

dichloromethane (DCM), dimethylamino pyridine (DMAP), N-(3-dimethylaminopropyl)-N′-

ethylcarbodiimide hydrochloride (EDCI), copper bromide CuBr, N,N,N′,N′′,N′′-

pentamethyldiethylenetriamine (PMDTA), 5-hexynoic acid, hydrochloric acid, and

poly(ethylene oxide) monomethyl ether Mn = 750 g/mol were used as obtained. Novozym 435

(derived from Candida antarctica type B and immobilized on an acrylic macroporous resin)

was dried under vacuum at 4 °C over P2O5 for two days prior to use. DVA was purchased

from TCI-Europe. 3-Methyl-3-oxetanemethanol was purchased from Alfa Aesar. The

membranes used for dialysis were bought from Spectrum Laboratories (regenerated cellulose)

and had an MWCO of 1000 g/mol.

2.2.2 Measurements

Weight-average molar mass (Mw), number-average molar mass (Mn), and molar mass

distribution (Mw/Mn) were measured by gel permeation chromatography (GPC) Viscotek

GPCmax VE 200 using DMF or THF as eluent with a flow rate of 1 mL/min through column

set HHR + GMHHR-N (Viscotek, mixed bed). The GPC was equipped with a refractive index

detector (VE 3580 RI detector, Viscotek). Polystyrene standards were used for calibration. In

the case of using THF, the temperature of the column was adjusted to 22 and to 60 °C for

DMF.


18

1H NMR and

13C NMR spectra were recorded using a Varian Gemini 2000

spectrometer operating at 400 MHz for 1H NMR and 200 MHz for

13C NMR spectroscopy.

The surface tension γ of the aqueous polymer solutions was measured by the Wilhelmy

plate method using an automated DCAT tensiometer (Data Physics Instruments). A solution

of 0.036 g/L of the polymer in bidistilled water was filtered through a 0.45 μm pore-size

PTFE filter prior to use. Following each injection, the surface tension was measured after 10

min of stirring and a 2 h waiting period. Measurements were carried out at 25 °C.

DLS measurements were performed using an ALV/DLS-5000 instrument (ALV

GmbH). As a light source a 20 mW He−Ne gas laser was used (Uniphase, 632.8 nm). The

DLS instrument was equipped with a goniometer for automatic measurements between

scattering angles θ of 30 and 140°. The correlation functions were analyzed by the CONTIN

method, which gives information on the distribution of decay rate (Γ). Apparent diffusion

coefficients were obtained from Dapp = Γ/q2(where q = (4πn/λ) sin(θ/2), λ is the wavelength of

the light, n is the refractive index, and θ is the scattering angle). Finally, apparent

hydrodynamic radii were calculated via Stokes−Einstein equation. The polymers were

dissolved in bidistilled water at a concentration of 1.25 g/L and directly filtered into the light

scattering cells through a 0.45 μm pore size PTFE filter. The hydrodynamic radii were

determined at 10 to 12 different angles and averaged for each concentration.

The surface pressure (π) as a function of mean molecular area (mmA) was measured

using a Langmuir trough system (KSV, Helsinki, Finland) with a Teflon trough and a

microroughened platinum Wilhelmy plate. The temperature of the water of the subphase was

maintained at 20 °C. The compression and expansion rate for all experiments was 750

mm2/min. In the case of relaxation experiments, after expansion, a waiting period of 20 min

was included.

2.2.3 Preparation of 2-(azidomethyl)-2-methylpropane-1,3-diol (AMD)

2-(Bromomethyl)-2-methylpropane-1,3-diol was synthesized according to the procedure

described by Lugo-Mas et al.130

For AMD synthesis, a mixture of 2-(bromomethyl)-2-

methylpropane-1,3-diol (5 g, 27.3 mmol), sodium azide (7.1 g, 109 mmol), and 100 mL of

DMSO was added to a 250 mL round-bottomed two-necked flask. The mixture was stirred for

2 days at 80 °C. Then, DMSO was removed at 80 °C by rotary evaporation under reduced

pressure. The organic residue was cooled using an ice bath. Distilled water (50 mL) was

added gradually under stirring, and the solution was extracted three times using DCM. The

organic phase was dried overnight using magnesium sulfate. The solvent was removed under

reduced pressure. The synthetic route to prepare this monomer is revealed in Figure 2.1. The


19

NMR data were as follows. 1H NMR (400 MHz, CDCl3) δ = 3.60 (d, 4H, 2 CH2OH), 3.45 (s,

2H, N3−CH2), 2.26 (s, 2H, 2 −OH), 0.86 (s, 3H, −CH3). 13

C NMR (200 MHz, CDCl3) δ =

68.08 (CH2−OH), 55.56 (CH2−N3), 40.93 (C), 17.52 (−CH3).

Figure 2.1 Synthesis of 2-(azidomethyl)-2-methylpropane-1,3-diol

2.2.4 Typical enzymatic polycondensation procedure

A mixture of 2-(azidomethyl)-2-methylpropane-1,3-diol (713 mg, 4.9 mmol), DVA (974

mg, 4.9 mmol), and CAL-B (61 mg) was added to a Schlenk tube, and the mixture was stirred

under nitrogen at 60 °C for 3 days. Then, the reaction was quenched by the addition of ~30

mL of DCM, followed by filtration to remove the acrylate beads carrying the enzyme. The

organic layer was washed with distilled water three times and then dried using magnesium

sulfate overnight. The solvent was removed under reduced pressure. The polymer was

precipitated from THF into n-hexane and dried. The resulting polymer is called PAA. Mn was

3100 g/mol and Mw/Mn was 1.6. 1H NMR (400 MHz, CDCl3) δ = 4.43 (s, 1H, −OH), 3.96 (s,

2H, 2C−CH2−O), 3.43 (s, 2H, N3−CH2−C at the end group), 3.34 (s, 2H, N3−CH2−C), 2.36 (s,

4H, 2CH2−CO), 1.74 − 1.58 (m, 4H, 2CH2−CH2−CH2), 1.00 (s, 3H, CH3), 0.96 (m, 3H,

CH3 at the end group). PAA had a glass-transition temperature of about −43 °C without the

presence of any melting peak

2.2.5 Typical procedure for synthesis of poly(2-(azidomethyl)-2-methylpropane

adipate)-g- poly(ethylene oxide) (PAA-g-PEO)

Alkyne functional poly(ethylene oxide) monomethyl ether (alkyne-PEO) was synthesized

according to Gao et al.131

PAA (100 mg, 0.39 mmol with respect to azide groups) of Mn =

3100 g/mol, alkyne-PEO (363 mg, 0.429 mmol), and anhydrous THF (3 mL) were placed in a

Schlenk tube. The mixture was agitated using a magnetic stirrer and sealed using rubber

septum. Degassing was carried out by bubbling nitrogen for 15 min. This was followed by the

addition of 11 mg (0.08 mmol) CuBr and 0.018 mL (0.9 mmol) of PMDTA. The solution was

stirred for 24 h at room temperature. At the end, the reaction solution was diluted using ~30

mL of THF, followed by passing it through a silica gel column. The solvent was removed


20

using rotary evaporation at 40 °C. The residue was dissolved using 10 mL of distilled water,

followed by dialysis against distilled water for 4 days using regenerated cellulose membrane

with MWCO of 1000 g/mol. The polymer was freeze-dried to obtain PAA-g-PEO with yield

of 40%, Mn = 13750 g/mol, and Mw/Mn = 1.6. 1H NMR (400 MHz, D2O) δ = 7.68 (s, 1H,

triazol-CH-C), 4.33 (s, 2H, CH2-O-CH2−CH2−O-CO), 4.10 (s, 2H, C−CH2-triazol), 3.80 (d,

4H, 2 C−CH2−O-CO), 3.71 (m, 68H, O−CH2-CH2−O), 3.24 (s, 3H, O−CH3), 2.60 (s, 2H,

CH2−CH2−CH2−COO), 2.40 (m, 6H, 3 CH2−CH2−COO), 1.80 (s, 2H, triazol-C−CH2), 1.45

(s, 4H,2 CH2−CH2−COO), 0.81 (s,3H, C−CH3).

Figure 2.2 A) Enzymatic synthesis of polyester with pendant azide aroups (PAA) and

B) Grafting reaction to PAA using alkyne-PEO


21

2.2.6 Synthesis of PAA-g-PEO using enzymatic polymerization and “Click” chemistry

in one-pot process

2-(Azidomethyl)-2-methylpropane-1, 3-diol (285 mg, 1.97 mmol), DVA (390 mg, 1.97

mmol), and immobilized lipase CAL-B (14 mg) were charged into a Schlenk tube, and the

mixture was stirred for 3 days at 60 °C. At the end, a solution of alkyne-PEO (1.831 g, 2.17

mmol), CuBr (56 mg, 0.39 mmol), PMDTA (0.094 mL, 0.45 mmol), and 5 mL of anhydrous

THF was added to the Schlenk tube, which contained the polyester and the enzyme. The

mixture was then degassed by bubbling nitrogen for 15 min. The solution was stirred for 24 h

at room temperature, then diluted with THF and purified first by filtration to remove the

enzyme beads, followed by passing it through a silica gel column to remove the CuBr. The

solvent was removed by rotary evaporation at 40 °C under reduced pressure. Further

purification was carried out using dialysis against distilled water for four days using

regenerated cellulose membrane with MWCO of 1000 g/mol. The polymer was freeze-dried

to obtain PAA-g-PEOop with Mn = 11100 g/mol and polydispersity of Mw/Mn = 2.1. The 1H

NMR spectrum of this polymer shows the same peaks as the previously discussed graft

polymer.

2.3 Results and discussion

2.3.1 Enzymatic polycondensation of poly(2-(azidomethyl)-2-methylpropane adipate)

(PAA).

Figure 2.2 shows the enzymatic polycondensation of DVA with AMD, which yields

poly(2-(azidomethyl)-2-methylpropane adipate) (PAA). Table 1.1 summarizes the

polycondensation results using different conditions. As described above, the polymer is

synthesized using CAL-B as catalyst, and it is generally considered that polycondensation of

diols and activated esters does not occur in the absence of enzyme in the temperature range up

to 60 °C,132

which is also confirmed by our results. All polycondensation of DVA with AMD

show a significantly lower activity compared with the use of DVA and glycerol. This is

indicated when doing the polycondensation under identical conditions, as described in

literature, but replacing glycerol by AMD.124

This can be explained by (i) the increased steric

hindrance when the substituents at the C2 carbon of glycerol (H and OH) are replaced by

CH3 and CH2N3 in AMD and (ii) the fact that AMD is a prochiral monomer. At the moment,

there is no comprehensive view on the influence of substituents on the reaction kinetics of


22

enzymatic polycondensation. Peeters et al133

studied the influence of sterical hindrance in the

ROP of 4-substituted caprolactones. It was shown that deacylation becomes rate-determining

upon increasing the substituent size. This fact might play the same role in enzymatic

polycondensation reaction. Mahapatro et al. found that using monomers as diacids and diols

having more CH2 groups in lipase-catalyzed polycondensation results in higher molar mass

polyesters compared with shorter chain-length species.134

Table 1.1 Enzymatic polycondensation of DVA and AMD.

Condition Temperature

(°C)

Time

(days)

Mn1)

(g/mol)

Mw/Mn

bulk 50 3 2,000 1.7

bulk 60 3 3,100 1.6

bulk 90 3 2,200 1.9

Toluene2)

60 3 2,100

1.8

bulk3)

60 3 ---

---

1) Mn and Mw/Mn results were obtained by GPC.

2) The concentrations of DVA and AMD were 1.97 mol/L.

3) The experiment was performed in the absence of CAL-B.

For bulk polycondensation at different temperatures and keeping all other parameters

constant, the highest Mn value is obtained at 60 °C. These results are in agreement with

enzymatic ring-opening polymerization of ε-caprolactone at different temperatures. Also, in

this case an increase in Mn with temperature is observed until 60 °C, followed by a decrease at

higher temperatures.135

For another lipase-catalyzed polycondensation, the highest Mn value

is obtained at 50 °C.136

For comparison, one polycondensation is carried out using toluene as

a solvent because a higher activity can be achieved by CAL-B in ring-opening polymerization

of ε-caprolactone when using toluene as solvent instead of bulk conditions.135

This is different

for the system under consideration where the enzyme shows more activity when performing


23

the reaction in bulk compared with solution polycondensation in toluene. The PAA samples

are characterized by 1H NMR spectroscopy as shown in Figure 2.3.

All peaks can be assigned to the structure of PAA. A minor side reaction indicated by the

peaks z, u, and v is observed, which is most prominent at a reaction temperature of 90 °C. The

side reaction occurs between vinyl end groups and azide groups.

Figure 2.3 1H NMR spectra of PAA synthesized at 60 °C and at 90 °C in CDCl3.

This type of reaction has been previously reported for activated olefins and

azides.137

The reaction is proposed to proceed according to Figure 2.4. First, an azide group

will react with a vinyl group according to Huisgen 1,3-dipolar cycloaddition,138

and second,

an elimination reaction139

occurs to form a 1,2,3-triazole ring and a carboxylic acid group.

The amount of repeating units that has a 1,2,3-triazole ring instead of an azide group is

calculated by the ratio between peaks u and d and is 1.4 mol % and 6.7 mol % for the PAA

synthesized at 60 and 90 °C respectively. Signals from vinyl end groups do not appear, which

indicates hydrolysis of these end groups during the polycondensation reaction.136


24

Figure 2.4 Proposed side reaction mechanism between vinyl ester end groups and pendant

azide groups during polycondensation reaction.

2.3.2 Synthesis of PAA-g-PEO using CuAAC “Click” chemistry

The synthesis of PAA-g-PEO is carried out by “grafting onto” strategy using CuAAC

“click” reaction. The reaction is performed under mild conditions in the presence of 10 mol %

excess of alkyne-PEO. Figure 2.2B shows the grafting reaction that is carried out in

anhydrous THF. This “click” reaction is fastest when using PMDTA as ligand to dissolve

CuBr.100

The GPC traces of Figure 2.5 show that PAA-g-PEO has a higher molar mass than

PAA, indicating the successful grafting reaction. Both traces have a small aggregation peak at

small retention volume. This peak disappears when THF is used as a solvent for GPC.

Figure 2.5 GPC traces of PAA and PAA-g-PEO in DMF at 60 °C.


25

Usually, the molar masses of PAA are determined by GPC employing THF.

Unfortunately, using THF for PAA-g-PEO results in nonsymmetric peaks in the GPC traces

because PEO chains obviously interact with the column material, which has been reported in

the literature.140

Figure 2.6 shows the 1H NMR spectrum of PAA-g-PEO in D2O. The ratio

between peak a and peak p is 3:2.9 which indicates that there is approximately one PEO chain

for each repeating unit.

Furthermore, Figure 2.7 depicts the complete disappearance of the vibration of the azide

group in the FT-IR spectrum at 2100 cm−1

as a result of the coupling reaction, which is also

an indication of quantitative reaction.

Figure 2.6 1H NMR spectrum of the PAA-g-PEO in D2O at room temperature.

Usually, in polymer analogous reactions, the polymer reactivity is sterically hindered

when the functional group is close to the polymer backbone.141

Even by using “click”

reactions for the “grafting onto” route, the reactions are not always quantitative.142

In fact, to

increase the density of graft chains on the polymer backbone, steric hindrance must be

lowered by increasing the spacer length between the repeating azide or alkyne units and the

polymer backbone and by using reactive oligomers to be grafted with less bulky structures,

such as, for example, PEO. Also, an excess of the oligomer grafted to the backbone improves

the grafting density.131

Additionally, ligand, solvent, and temperature also affect the yield of


26

the click reaction.100

In the case under investigation, only one CH2 group separates the azide

group and the polymer backbone. Nevertheless, the grafting reaction is quantitative.

Figure 2.7 FT-IR spectra of PAA and PAA-g-PEO.

2.3.3 Synthesis of PAA-g-PEO in sequential one-pot reaction (PAA-g-PEOop)

The graft copolymer is also synthesized by polycondensation of DVA and AMD in the

presence of the CAL-B as catalyst, followed by the addition of alkyne-PEO, ligand, CuBr,

and the solvent to the same pot. The solution then undergoes bubbling with nitrogen for 15

min. Carrying out the chemical reactions in one pot has the advantage of accelerating the

synthetic procedures by reducing the number of purification steps, therefore leading to more

ecofriendly products.143 The reaction must be carried out sequentially for two reasons. First,

the presence of copper ions in the reaction vessel can inhibit the lipase during

polycondensation.144 Second, any AMD converted with alkyne-PEO might be unable to

undergo enzymatic polycondensation with DVA because of steric hindrance caused by the

attached PEO chains. One-pot lipase-catalyzed ring-opening polymerization and ATRP

polymerization in the presence of CuBr as catalyst for ATRP polymerization was carried out

successfully in one step but by using super critical carbon dioxide as a solvent. Other solvents

suitable for this procedure could not be identified.145,146 Both 1H NMR and FT-IR spectra of


27

the PAA-g-PEOop are identical to that of PPA-g-PEO, which means that the reaction also

performs quantitatively, but as shown in Figure 2.8, when the GPC traces of the two polymers

are compared, it is obvious that the PAA-g-PEOop has a broader molar mass distribution than

PAA-g-PEO, simultaneously, the sequential one-pot synthesis leads to a polymer with a

smaller molar mass.

Figure 2.8 FT-IR spectra of PAA and PAA-g-PEO.

The reason for this difference is the fact that PAA-g-PEO is synthesized from PAA

without oligomers because it is purified prior to grafting. In the case of PAA-g-PEOop, the

click reaction was carried out directly after the polycondensation without carrying-out any

purification step, which means the remaining oligomers will react with PEO to yield a larger

number of graft copolymers with lower molar mass.

2.3.4 Surface tension measurements

The surface tensions γ of aqueous solutions of PAA-g-PEO are measured as a function of

polymer concentrations at 25°. Plotting γ versus polymer concentration (log C) yields the

critical aggregation concentration (cac) indicated by the intersection of the extrapolation of

the two linear regimes where the curve shows an abrupt change in slope. (See Figure 2.9) The

value obtained by this method is surprisingly low at 3 × 10−2

μM.

Amphiphilic graft and brush copolymers usually have low cac values.147

This value is

lower than that for conventional surfactants and block copolymers.110

Small cac values will


28

strongly nominate the polymer for drug delivery application because the in vivo stability after

injection is improved.107

Figure 2.9 Surface tension of PAA-g-PEO in water as a function of polymer concentration

at 25 °C.

2.3.5 1H NMR spectroscopy in water and in THF

THF is a good solvent for the polyester backbone and the grafted PEO chains. For that

reason, a comparison between the 1H NMR spectra of PAA-g-PEO in water (which is a

nonsolvent for the polymer backbone but a good solvent for PEO) and THF is carried out.

(See Figure 2.10.) Actually, a broadening of the polyester backbone peaks in the spectrum

obtained in D2O can be recognized easily in contrast with the same peaks in the spectrum

obtained in THF-d8. Such broadening is the result of a decreased mobility of protons of the

polymer chains with hindered motion.148

In fact, the 1H NMR spectrum of PAA-g-PEO in

D2O confirms the formation of large polymer aggregates in water, as will be discussed below.

http://pubs.acs.org/doi/full/10.1021/bm1011085#fig7


29

Figure 2.10 Comparison between the 1H NMR spectra of PAA-g-PEO in D2O and in

THF-d8.

2.3.6 Dynamic light scattering

In DLS measurements of PAA-g-PEO in water, for all angles and concentrations above

cac, two different species have always been observed. The corresponding hydrodynamic radii

can be attributed to single chains with a typical value of 6 nm and larger aggregates of 75 nm.

Figure 2.11 shows the hydrodynamic radius Rh distribution obtained at the polymer

concentration of 1.25 g/L and scattering angle θ = 80° measured at 25 °C. Furthermore, the

average hydrodynamics radii for both species at different polymer concentrations are

depicted. The error bars indicate the standard deviation of the averaging over all

measurements at different angles. No significant increase in aggregate size can be observed

with increasing polymer concentration. The hydrodynamic radii of both species vary only

weakly with concentration.


30

Figure 2.11 Hydrodynamic radius distribution of PAA-g-PEO in water at a concentration

of 1.25 g/L, scattering angle of 80°, and temperature of 25 °C. The inset

shows the concentration dependence of Rh for the two species identified by

DLS.

2.3.7 Langmuir trough measurements

Figure 2.12 shows the Langmuir isotherm of PAA (Mn = 3100 g/mol) at the air/water

interface. The isotherm shows a horizontal region at a surface pressure π of 13 mN/m without

having a further increase prior to final collapse. This indicates a weak anchoring of the

molecules at the air−water interface. Two different mechanisms can occur in this case: One is

according to a gradual formation of “giant folds” or “multiple folds” with an extension into

the subphase.149

The other mechanism is according to a multilayer formation of the PAA on

the water surface.150,151

To investigate the mechanism, a reversibility experiment for PAA is

performed by compression−expansion steps. In the case of PAA, no significant differences of

the isotherms can be observed. This indicates that all chains remain flexible and stay at the

water surface. The isotherm of PAA-g-PEO shows a typical isotherm for amphiphilic block or

graft copolymers that contain PEO as water-soluble part.152,153

The isotherm has a significant

pseudoplateau indicating the phase transition from pancake to brush.154

At the end of the

plateau, the hydrophilic chains form brush domains, and the hydrophobic parts anchor them to

the water surface. Typically, the surface pressure at which the pseudoplateau appears for


31

grafted copolymers is 16 mN/m slightly larger than that in the case of PEO block copolymers

(between 9 and 13 mN/m depending on the length of PEO chains).155,156

With further

compression, the surface pressure increases again until at 29 mN/m the slope of the isotherm

changes significantly, which can be assigned to the monolayer collapse. To investigate the

mechanism of this collapse, a reversibility experiment is performed. Because of the limited

trough size, mmA values between 200 and 10 Å2 are chosen for this experiment to cover the

range between phase transition (pseudoplateau) and collapse region.

Figure 2.12 π-mmA isotherms of PAA and PAA-g-PEO at tthe air-water interface at

20°C.

During expansion, the surface pressure is lower compared with the compression curve at

identical mmA values, but the course is similar. During relaxation after expansion, the surface

pressure approaches the initial isotherm, and starting from the end of the pseudoplateau

during the following compression, the initial behavior is reproduced. This behavior can be

explained according to the long relaxation time needed for PEO chains to move from the

water subphase to the water surface. Beyond the end of the pseudoplateau, the hydrophobic

parts are dominating the shape of the isotherm, which are, as in case of neat PAA, fully

relaxed. The reproduction of the isotherm in this higher pressure regime indicates that no

chains are lost in the subphase during collapse. This suggests that the collapse point indicates


32

a multilayer formation. The PAA-g-PEO has a higher surface activity compared with PAA

because the PEO chains anchor the polymer backbone at the water surface effectively during

compression, resulting in a larger collapse pressure, but their hydrophilicity is not sufficient to

remove the PAA backbone of PAA-g-PEO from the air−water interface to the water

subphase.

2.4 Conclusions

Aliphatic polyesters with pendant azide groups were successively synthesized using

enzymatic polycondensation of 2-(azidomethyl)-2-methylpropane-1,3-diol and DVA. The

enzymatic reaction was performed at different temperatures. The highest molar masses were

obtained at 60 °C by polycondensation in bulk. A side reaction between azide groups and

vinyl end groups was observed to a minor extent, which becomes more pronounced at higher

polycondensation temperatures (90 °C). The mechanism suggested is based on the Huisgen

1,3-dipolar cycloaddition, followed by elimination reaction resulting in a 1,2,3-triazole ring.

The polyester was quantitatively modified using alkyne-PEO. The reaction to the

corresponding graft copolymer was quantitative. Both enzymatic polymerization and “click”

reaction were successfully carried out in sequential one-pot synthesis. Surface tension

measurements show that the graft copolymer had a very small cac. Above cac, stable

aggregates with a size of 75 nm are formed at different concentrations, as revealed by DLS.

The aggregate formation was obviously the result of the amphiphilic character of the graft

copolymer. The polyester backbone was hydrophobic, whereas the PEO graft chains were

hydrophilic. This was also confirmed by 1H NMR spectroscopy. Langmuir trough

experiments showed that the isotherm of the graft copolymer had a pancake-to-brush

transition. The PEO anchored the PAA onto the water subphase, and by relaxation

experiments, it was found that the PAA layer remains flexible at the surface, even after

monolayer collapse. The polyester backbone of the new polymer can be considered to be

biodegradable, and the remaining PEO parts have a sufficiently low molar mass, so the graft

copolymer can be excreted in vivo via kidneys. Therefore, advantages compared with the use

of poloxamers can be expected in the field of biomedical applications.157

Chapter 3 Poly(glycerol adipate) to Synthesize Graft Copolymer

33

Chapter 3

Utilization of Poly(glycerol adipate) to Synthesize Graft Copolymer and

Polymeric Analogues of Glycerides

3.1 Introduction

Synthetic polyesters are one of the most widely used polymers in modern life. Their

biodegradability and low cost of production are considered to be the main factors for their

wide global demand. Nevertheless, many physical, biological and mechanical properties of

polyesters are not always meeting the crucial requirements for some applications. Therefore,

synthesizing new polyesters with functional groups able for post-polymerization

functionalization is a challenging task.158

Many routs have been developed to synthetize

polyesters with functional groups such as ring opening polymerization of substituted

lactones,61,119,121,159

or by polycondensation of multifunctional monomers.62,64,80,160,161

Utilization of enzymes as a catalyst to synthesize functional polyesters has been attracting

many interests for two decades due to advantages that enzymes can provide over conventional

chemical catalysts.162

Enzymatic polymerization can be performed under mild reaction

conditions and does not require protection-deprotection steps due to the regio- and

chemoselectivity of enzymes.82

This will protect the polyester from possible degradation that

might occur to the polyester backbone during deprotection processes. Poly(glycerol adipate)

(PGA) was enzymatically synthesized first by Kline et al. 124

using glycerol and divinyl

adipate as monomers and lipase enzyme from Candida Antarctica type B (CAL-B) as catalyst

to yield linear polyesters with free pendent hydroxyl groups. The enzyme is immobilized on

an acrylic macroporous resin which facilitates the processes of separating it from the final

polymer. The overall synthesis process could be described as a simple, clean, easy to conduct,

easy to purify the final product, and easy to scale up to 500 g.163

Using glycerol as a monomer

has a big advantage since it is cheap, widely used, and biocompatible compound. On the

contrary, the utilization of divinyl adipate as monomer for synthesis is not appropriate for

commercial purposes. Using dimethyl adipate (DMA) instead of divinyl adipate to synthesize

poly(glycerol adipate) (PGA) is more appropriate since it is a large-scale and cheap

commodity chemical compound. In the previous chapter, a graft copolymer was synthesized

by utilization of a polyester called PAA which has free pendant azide group on every

monomer unit. Utilization of PGA instead of PAA to synthesize identical graft copolymers

seems reasonable for both commercial and environment-friendly purposes. The conversion of

glycerol with different fatty acids may result in mono-, di- or triglycerides.140

Triglycerides


34

are usually hydrophobic and the mono- or diglycerides can be considered as amphiphilic

molecules due to remaining OH-groups.164

Thus, they are surface active and reduce the

surface tension of water or they are effective emulsifiers.165

Additionally, they are able to

form lyotropic liquid crystalline phases.166

Especially, the monoolein/water system is

interesting since it can form cubosomes when stabilized by poloxamers.167

These

nanoparticles can dissolve both hydrophilic and lipophilic drugs and additionally they can be

used as scaffold for therapeutic proteins.168

It is reasonable to assume that polyesters based on

glycerol can achieve similar properties as low-molar mass glycerides discussed above. They

would have the advantage of higher mechanical stability and longer in vivo circulation times.

Esterification of the hydroxyl pendant groups at the PGA backbone with fatty acids has been

found to yield promising materials for application in the field of nano-drug carriers.122,169–171

However, further characterization for these polymers and corresponding nanoparticles is

necessary in order to tune their properties and to ensure their ability to be applicable in vivo.

Furthermore, additional applications in pharmacy and medicine can be envisioned due to

biocompatibility and biodegradability.


3.2.1 Materials

All chemicals were purchased from Sigma-Aldrich and used without further purification

unless otherwise stated. Lauroyl chloride 98%, stearoyl chloride 97%, behenoyl chloride

≥99%, copper bromide 99.99%, N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA),

dichloromethane ≥99.5% (DCM), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

hydrochloride (EDC), 4-(dimethylamino)pyridine (DMAP) dimethyl adipate (DMA) (99.5%)

are used as received. Tetrahydrofurane is dried over sodium under anaerobic conditions.

Pyridine (99%) was dried over calcium hydride overnight, distilled under atmospheric

pressure and stored over molecular sieve (3Å). Solvents for column chromatography and

precipitation were distilled prior to use. CAL B (lipase B from Candida Antarctica

immobilized on an acrylic macroporous resin) is dried under vacuum at 4 °C over P2O5 for

two days prior to use. Divinyl adipate (DVA) is obtained from TCI-Europe. The synthesis of

azide-terminal poly(ethylene oxide) monomethylether (mPEO-N3) was performed as

described by Gao et al.31

The number-average molar mass was Mn = 2000 g/mol


35

3.2.2 Synthesis of poly(glycerol adipate) (PGA)

Ploy(glycerol-adipate) was synthesized at 50°C from glycerol and DVA as described by

Kallinteri et al.122

A typical procedure for the synthesis of PGA backbone using DMA and

glycerol as monomers was as follows: (11.5 g, 0.12 mol) glycerol, (21.7g, 0.12mol) DMA,

and 13 ml anhydrous THF were charged into an oven dried two-necked 250 ml round bottom

flask.

Figure 3.1 Synthesis of (a) poly(glycerol adipate) backbone by using glycerol and either

DVA or DMA as monomers and CAL-B as a catalyst. (b) Esterification reaction

to obtain PGA-alkyne, (c) “Click” reaction to synthesize PGA-g-PEO.(d)

Esterification reaction between acyl chloride of fatty acids.


36

The flask was equipped on the top with soxhlet extractor (150 ml) attached to a condenser.

The soxhlet extractor was charged with 105 g of molecular sieve 5Å and then filled with

about 100 ml anhydrous THF. The mixture was stirred by magnetic stirrer to allow reactants

to warm up to the bath’s temperature (60°C) for about 30 min. The reaction was started by

adding (0.73 g) of enzyme. The pressure was then reduced gradually to 300 mbar in order to

allow for the evaporation process. The azeotropic mixture of THF and methanol was collected

gradually into the soxhlet extractor and became into contact with the molecular sieve. As a

result the methanol was entrapped gradually by the molecular sieve. The conditions

(temperature and pressure) were adjusted to allow one cycle of soxhlet filling to be 20 min.

The enzyme was removed at the end of polymerization by filtration followed by washing with

35 ml THF. The solvent was removed by rotary evaporation at 60°C under vacuum. Finally,

the temperature was raised up to 95°C for 10 min in order to deactivate any remaining free

enzyme. The polymer was used for the next step without further purification.

3.2.3 Acylation of PGA backbone with fatty acid chains

Acylation reaction is carried out between hydroxyl groups on PGA backbone and acyl halide

of lauroyl, stearoyl and behenoyl acids. The acylation reaction is performed also using the

procedure described by Kallinteri et al.122

However, further purification step is necessary in

order to remove unreacted fatty acid. The purification is carried out by precipitation into cold

n-hexane in the case of acylation with lauroyl chloride or for low substitution degrees in the

case of the other fatty acids. Whereas, dialysis against THF for 5 days, using regenerated

cellulose membrane of a MWCO of 1000 g/mol, is performed in case of higher substitution

degrees. PGA with molar mass 3700 g/mol is used for acylation reaction. The acylation

degrees (given in mol% of converted OH-groups of PGA) were as the following:

lauroyl chains: 30%, 50%, 75% called L30, L50 L75.

stearoyl chains: 8%, 20%, 45%, 65%, 85% called S8, S20, S45, S65, S85.

behenoyl chains: 45%, 65% called B45, B65

3.2.4 Synthesis of alkyne modified poly(glycerol adipate) (PGA-Alkyne).

A solution of PGA (1.5 g, 7.41 mmol with respect to OH groups), 5-hexynoic acid (2.1 mL,

18.5 mmol) dissolved into 20 mL DCM were added to an oven dried 100 mL two-necked

round bottom flask. The mixture was cooled with an ice bath. Afterwards, DMAP (293 mg,

0.96 mmol) and DCC (3.44mL, 14.85 mmol) dissolved into 10 mL of DCM were dropwise

added to the polymer solution over 20 min. The mixture was stirred at the room temperature

for 24 h to yield a brownish solution. The solution was then filtered to remove the resulting


37

precipitate. The organic solution was then extracted three times using distilled water. The

resulting organic layer was dried by anhydrous sodium sulfate. The polymer was further

purified by precipitation into cold n-hexane two times followed by drying at 40°C.

3.2.5 Synthesis of poly(glycerol adipate)-g-poly(ethylene oxide) PGA-g-PEO.

PGA-alkyne (150 mg, 0.51 mmol with respect to alkyne groups) and azide-terminated

poly(ethylene oxide) monomethylether (1.2 mg, Mn=2000 g/mol, 0.56 mmol) were dissolved

in 3.5 mL anhydrous DMF and then added to a 10 mL oven dried Schlenk flask. The mixture

was agitated using magnetic stirrer and sealed using rubber septum. Degassing was carried

out by bubbling nitrogen for 15 min. This was followed by addition of CuBr (21 mg, 0.15

mmol) and PMDETA (0.031 mL, 0.15 mmol). Further degassing was carried out using

nitrogen for 10 min. The solution was kept for 37 h at room temperature. The reaction was

quenched by open the rubber septum for 30 min. The solution was then diluted using THF,

and purified using a silica gel column to remove copper bromide. This was followed by

removing solvent using rotary evaporation at 40 °C under vacuum. The residue was

redissolved using 10 mL methanol followed by dialysis against water for four days using

regenerated cellulose membrane MWCO= 3500 Da. The polymer was finally dried using

freeze drying.

3.2.6 Polymer nanoparticle preparation

Nanoparticles are prepared according to the optimized interfacial deposition method.47

Shortly, 10 mg of polymer were dissolved in 1 mL of acetone which is then injected slowly

into 15 mL of 60 °C water using a glass syringe under rapid magnetic stirring. The hot

nanoparticle dispersion is subsequently poured into an empty iced beaker under magnetic

stirring. The remaining solvent and some water are then removed by a rotary evaporator to

obtain finally an 1% dispersion (10 mg polymer/g solution).

3.2.7 Differential scanning calorimetry

Differential scanning calorimetry (DSC) experiments are carried under continuous nitrogen

flow using a Mettler Toledo DSC 823e module. Aluminum pans were filled with about 10 mg

of sample. Every sample is heated up to 100 °C and kept at this temperature for 20 min. The

sample is then cooled until -50°C with a cooling rate of -1 K/min. The sample is kept at -50°C

for further 20 min, afterwards the sample is heated up again to 100°C with a heating rate 1

K/min. DSC traces were baseline-corrected. The maximum of the endothermal peak during

the second heating is taken as the melting temperature Tm, whereas the minimum of the


38

exothermal peak is taken as a crystallization temperature Tc. Specific enthalpy of melting

is obtained from integration of the endothermal peak divided by the weight of alkyl side

chains of the sample. The degree of crystallization XDSC= / where

is the

enthalpy of melting of the respective fatty acid.

3.2.8 Transmission electron microscopy (TEM)

The samples are negatively stained using aqueous solution of uranyl acetate. The samples for

freeze-fracture were cryofixed using a propane jet-freeze device JFD 030 (BAL-TEC,

Balzers, Liechtenstein). Thereafter, the samples were freeze-fractured at −150 °C without

etching with a freeze-fracture/freeze-etching system BAF 060 (BAL-TEC). Cryo-TEM grids

were prepared in the same way as the TEM, measurements were carried out immediately after

preparation of the grids with a Zeiss 902 A microscope operating at 80 kV.

3.3 Results and Discussion

3.3.1 Synthesis of poly(glycerol adipate) (PGA) backbone

PGA is an amphiphilic, water insoluble, yellowish, and highly viscous polymer. The overall

synthesis route for the PGA backbone is shown in (Figure 3.1a). The polymer is

enzymatically synthesized using two strategies. In the first one glycerol is enzymatically

polymerized with DVA in the presence of CAL-B as catalyst.

Figure 3.2 Experimental setup to prepare poly(glycerol adipate) using DMA and glycerol

in the presence of CAL-B as a catalyst and THF as solvent.


39

The rigioselectivity of lipase towards primary alcohols of the glycerol will yield linear

polyesters with free pendent hydroxyl groups at its backbone. The absence of enzyme, on the

other hand, results in cross-linked products.172

The vinyl alcohol produced as by-product,

beside PGA during the enzymatic reaction, is directly converted into acetaldehyde by

tautomerization and will finally evaporate at the reaction temperature. This will shift the

equilibrium of the polycondensation reaction towards the products. The second strategy to

synthesize PGA is using DMA instead of DVA. Utilization of DMA will cause some

problems of shifting the direction of the equilibrium towards the polymer since methanol will

be the by-product of this reaction which has to be removed. Actually, methanol and

tetrahydrofurane (THF) form an azeotropic mixture which makes their separation impossible

by distillation during the enzymatic reaction. Thus the polymerization reaction would be

ceased at low conversions. Therefore, the polymerization reaction is carried out in the

presence of molecular sieves placed into a soxhlet apparatus attached on the top of the

reaction vessel as depicted in Figure 3.2. Both methanol and THF evaporate together during

the enzymatic polymerization and condense again by the condenser to be collected finally into

soxhlet extractor where the mixture becomes in contact with the molecular sieve. The

molecular sieve has a pore size of 5Ǻ. This size will allow only methanol to be captured by

the molecular sieve and thus only pure THF will reflux to the reaction vessel. The capacity of

the molecular sieve to entrap methanol is around 14 wt% of its weight. An excess of about 80

wt% of molecular sieve is added in order to prevent the system to reach a state of saturation of

the molecular sieve with methanol. The procedure described above to remove the resulting

by-product has many advantages, e.g. easy to scale-up by increasing the amount of molecular

sieve and the molecular sieve is not in contact with the polymer formed. Many strategies have

been suggested to remove the resulting by-product in order to shift the reaction equilibrium

towards the products in polycondensation processes.173

However, not all of these strategies

are convenient for both laboratories and industrial applications.174–176

Using the solvent route

instead of bulk route for the polycondensation provides a better distribution for both enzyme

beads and temperature within the reaction vessel. Furthermore, Juais et al. 177

proved that

carrying out enzymatic polymerization in solvents gives a higher Mw than in bulk. The

procedure can be extended to be suitable for large scale processes. Table 3.1 shows the results

of enzymatic polymerization of divinyl adipate or dimethyl adipate with glycerol at different

reaction times. Increasing Mn of the polymer causes also an increase of its polydispersity D.


40

Table 3.1 SEC of PGA synthesized using either DVA or DMA and glycerol.

The number-average molecular weights Mn obtained from the reaction of DVA are higher

than that of DMA within a shorter time of reaction. The small reactivity of alkyl esters

towards alcohols in lipase-catalyzed transesterification could be the reason for these results.82

Figure 3.3 1H-

13C COSY NMR spectrum of PGA measured in CDCl3 at room

temperature.

Kind of adipate Time [h] Mw

(g/mol)

Mn

(g/mol) D

Divinyl adipate 4 5,070 2,700 1.9

Divinyl adipate 8 7,650 3,500 2.1

Dimethyl adipate 18 1,660 890 1.8

Dimethyl adipate 48 4,800 1,950 2.4


41

PGA (Mn is 1950 g/mol) is characterized first by 1H-

13C COSY NMR carried out in CDCl3

and shown in Figure 3.3. The APT NMR spectrum reveals that the peaks at 51.1, 66.1, 68.6,

69.2, 71.9 ppm have a negative value which indicates that they are related to methine (CH)

and methyl (CH3) groups. The peak at 51.1 ppm is well known to be related to the methyl

group of dimethyl adipate. This essentially means that the other peaks are related to the

methine group of glyceride units within the PGA backbone. The peaks in the 1H-

13C COSY

NMR spectrum are assigned to the polymer structure as shown in Figure 3.3. The presence of

many peaks for methine groups indicates some imperfections of the enzyme regioselectivity

during polymerization towards primary alcohols. This imperfection causes the formation of

1,2-disubstituted and 1,2,3-trisubstituted glyceride units within the backbone whereas only

1,3-disubstituted and 1-substituted species should appear in the case of ideal regioselectivity

of the enzyme during polymerization. The presence of some imperfections of the

regioselectivity has been noticed before.124,178

Actually, the presence of 1,2,3-trisubstituted

glyceride has the worst effect on the properties of the backbone since it decreases the number

of hydroxyl groups on PGA backbone and will effect also the linearity of the total polymer

backbone. The ratio of trisubstitution is calculated using the integral ratio between the peaks

R or S and the peak C and it is equal to about 8 mol%.

3.3.2 Temperature dependence of rigioselectivity

Two enzymatic reactions are carried between divinyl adipate and glycerol but at different

temperatures (60°C and 40°C) in order to investigate the influence of reaction temperature on

the rigioselectivity of the CAL-B. The corresponding 13

C NMR spectra of both reaction

products are shown in Figure 3.4. The comparison between both spectra shows a complete

disappearance of the peaks related to 1,2-disubstituted and 1,2,3-trisubstituted glycerides for

the polymer synthesized at 40°C. This indicates a perfect rigioselectivity of the enzyme at

40°C. These results are matching the results reported before for the enzymatic polymerization

of divinyl sebacate with glycerol at different temperatures.179


42

Figure 3.4 Expanded 13

C NMR spectra of PGA obtained from glycerol and DVA at

40°C, and at 60°C.

3.3.3 Synthesis poly(glycerol adipate)-g-poly(ethylene oxide) (PGA-g-PEO)

The synthetic pathway for PGA-g-PEO from PGA is shown in (Figure 3.1a and b). Alkyne

groups are introduced to the PGA backbone by the esterification reaction between the

hydroxyl groups of the PGA backbone and 5-hexynoic acid in the presence of DMAP and

EDCI as a catalyst. The 1H NMR spectrum of PGA-alkyne in CDCl3 is given in (Figure 3.5a).

The integral ratio between peak a and peak d is 4 : 0.97. This reveals nearly quantitative

coupling. The synthesis of PGA17-g-PEO44 is carried out under mild conditions in order to

prevent any type of degradation to the polymer backbone. Actually, an excess of 1.1 eq (with

respect to alkyne groups) of mPEO-N3 is added. Additionally, (Figure 3.5b) shows the 1H

NMR spectrum of PGA17-g-PEO44 in CDCl3. The grafting efficiency is calculated by the

integration of the ratio between peak w and peak d which is 3 : 0.97. Thus, the reaction is

quantitative within the experimental error. Furthermore, the comparison between the FT-IR

spectra of PGA17-alkyne and PGA17-g-PEO44 reveals the complete disappearance of the

alkyne vibrations of PGA17-alkyne at 650, 2115, and 3290 cm-1

as a result of the coupling

reaction as seen in Figure 3.6. The resulting PGA17-g-PEO44 is water soluble. The polymer

structure is similar to the structure of a graft copolymer synthesized using DVA and azide

functional diols followed by “click” chemistry.62


43

Figure 3.5 1H NMR spectrum of (a) PGA17-alkyne, (b) PGA17-g-PEO44 in CDCl3 at room

temperature and 400 MHz.

Figure 3.6 FT.IR spectra of PGA17-alkyne and PGA16-g-PEO17. Shaded areas represent

alkyne peaks.


44

3.3.4 Modification of PGA backbone with fatty acids

PGA backbone was acylated with aliphatic acid chlorides of lauric acid, stearic acid and

behenic acid. Pyridine was added to polymer solution as acid scavenger. The molar mass of

polymer backbone was estimated using SEC using THF as eluent and poly(styrene) as a

standard. The absence of unreacted fatty acid chains was confirmed by mono-modal

distribution of their SEC traces. The ratio of esterification and total molar mass of the

modified polymers were determined using the corresponding 1H NMR spectrum.

Figure 3.7 1H NMR spectra (400 MHz, CDCl3) of the comb-like derivatives of PGA with

stearoyl side-groups with esterification degrees from 20 mol% (PGA-S20) to

85 mol% (PGA-S85). Peaks and protons used for the calculation of the

esterification degree are indicated.

The degree of esterification of OH-groups of poly(glycerol adipate) was calculated from the

integrals of the peaks indicated in the 1H NMR spectra according to the equation:

Esterification degree (%) = [1.33×E1/(E2-(0.67×E1))]×100


45

3.3.5 DSC Measurements

Table II shows the results obtained by DSC. The PGA backbone is an amorphous polymer

which means that the crystallization of the substituted polymer is related only to the alkyl side

chains. The results reflect an increase of Tm and with increasing degree of substitution

and with increasing length of alkyl side chains.

Table 3.1 Melting temperature Tm, crystallization temperature Tc, specific enthalpy of

melting , degree of crystallinity XDSC

a No DCS peak is present under the measurements conditions.

The increase of Tm and with increasing side chain length is mainly related to the thicker

crystalline lamellae, which causes an increase of the energy required for melting the

polymer.180 The decrease of Tm with decreasing degree of substitution must be related to

thinner or more imperfect crystals, whereas the decrease in indicates also a higher

amount of amorphous alkyl chains. The results show also a decrease of the degree of

Sample Tm (°C) Tc

(°C) (J/g) XDSC (%)

L30 -37 --a

16.4 8.4

L50 -22 -25 61.3 26.4

L75 -20.6 -32 56.8 29.1

Lauric acid 45.3 -41 195.2 100

S8 29.1 21 34 14.6

S20 33.9 29.7 29.9 12.8

S45 36.9 35.4 100.7 43.2

S65 39 36.9 159.7 68.5

S85 38.7 34.8 162.9 69.9

Stearic acid 69.7 66.6 233 100

B45 57.9 57.3 145.2 59.6

B65 51.9 51.18 158.1 64.9

Behenic acid 80.1 76.1 243.7 100


46

crystallinity with decreasing degree of substitution. Nevertheless, the degree of crystallinity is

always lower than the crystallinity of the free fatty with identical chain length alone.

3.3.6 Thermogravimetry

The thermal decomposition temperatures of the stearic acid, PGA, S20, and S45 are

investigated using thermo-gravimetric analyses in an air stream with a heating rate of

5°C/min. The results are shown in Figure 3.8. The comparison between the thermograms

shows that the stability of S45 is higher than stearic acid or PGA. On the other hand, no big

effect of substitution on the stability of the overall polymer is noticed in the case of S20 which

has a low degree of substitution.

Figure 3.8 Thermal gravimetric analysis (TGA) thermograms of stearic acid, PGA, S20,

and S45 using a heating rate of 5 K/min in air.

3.3.7 Transmission electron microscopy

It has been reported by Kallinteri et al.122

that PGA substituted with stearoyl chains forms

spherical nanoparticles. However, our investigation shows that the shape of these

nanoparticles is strongly depend on the ratio of substitution. The cryo-TEM and negative


47

stain-TEM images (Figure 3.9a,b) show various non- spherical shapes of nanoparticles with

linear boarders and defined geometries such as hexagons, pentagons, squares, and triangles.

Figure 3.9 (a) Cryo-TEM image of S20 nanoparticles prepared in aqueous suspension.

(b) Negative-stain TEM of S20 nanoparticles. (d) Internal structure of S85

nanoparticles after freeze fracture showing a layered morphology.

Further investigations into these nanoparticles show the presence of well-ordered pseudo-

hexagonal structures.46

A freeze fracture image of S85 (Figure 3.9d) reveals the presence of

internal lamellar structure inside the nanoparticles which is explained as onion-like

morphology. Such onion structure is suggested to be as results of alternating crystalline and

amorphous phases within the body of the nanoparticles.46

3.4 Conclusions

This report describes the synthesis and characterization of poly(glycerol adipate) (PGA),

PGA-g-PEO and fatty acid modified poly(glycerol adipate). PGA is synthesized by enzymatic

polymerization using glycerol and either divinyl adipate or dimethyl adipate. Methanol which

is produced as a by-product during the enzymatic polymerization in the case of using DMA is

removed by molecular sieves packed into a soxhlet extractor on the top of the reaction vessel.

DMA shows a slower enzymatic polymerization rate compared with DVA. The


48

rigioselectivity of CAL-B is affected by reaction temperature. It has been found that comb-

like PGAs with branching are produced when the enzymatic polymerization is carried out at

60°C because of the rigioselectivity imperfection of CAL-B at this temperature. On the other

hand, linear PGA can be produced when the reaction is carried out at 40 °C. The OH pendent

groups on PGA were quantitatively attached to alkyne groups by esterifying them with 5-

hexynoic acid to get PGA-Alkyne. Azide-terminated poly(ethylene oxide) monomethylether

was grafted onto PGA-Alkyne by "click" reaction at room temperature to yield PGA-g-PEO.

The synthetic route described here is considered as alternative strategy to synthesize graft

copolymers similar to PAA-g-PEO as described in the previous chapter.

In the case of fatty acid modified PGA, both Tm, increase with increasing the ratio of

substitution and/or by increasing the length of alkyl side chains. This is explained as a result

of improvement of the compact packing of the complete comb-like polymers. Substitution is

also found to increase the stability of the polymers against thermal decomposition.

Transmission electron microscopy images of the nanoparticles depict shapes of the resulting

nanoparticles depending on the degree of substitution. S20 is found to form nanoparticles with

linear boarders with polyhedral geometries whereas S85 forms onion-like spherical

nanoparticles.

Chapter 4 Graft Copolymers Able to Form Worm-Like Aggregates

49

Chapter 4

Synthesis and characterization of graft copolymers able to form

polymersomes and worm-like aggregates

4.1 Introduction

Graft copolymers have attracted interest of polymer scientists over several decades due to

their existing commercial and potential applications.110,181

Actually, a strong tendency has

appeared to use them in the field of drug delivery instead of classic block copolymers due to

some superior properties.107,108

Biodegradability and biocompatibility of any polymer play an

important role when used for drug delivery systems. Poly(ethylene oxide) (PEO) is the

synthetic standard polymer in the field of pharmacy and medicine due to such properties as

e.g. low costs, water-solubility, biocompatibility.182

PEO is mainly attached to hydrophobic

polymers, proteins, and DNA in order to form a water coordination sphere protecting them

from being recognized by the immune system thus prolonging the in vivo circulation time.111

Introducing oligo-PEO (sometimes called) as graft segments to a hydrophobic polymer chain

can yield thermo-responsive polymers.183,184

Furthermore, such type of comb-like polymer

can form stable micelles with smaller cmc and melting point Tm compared to conventional

amphiphilic block copolymers.110

Using aliphatic polyesters as hydrophobic polymer

backbone is an elegant strategy to obtain graft copolymers that are candidates for

pharmaceutical applications.48,159

Poly(glycerol adipate) (PGA) is synthesized by enzymatic

polymerization of glycerol and divinyl adipate (DVA) to yield aliphatic linear polyesters with

pendent free hydroxyl groups.124

The process becomes even cheaper when DVA is replaced

by dimethyl adipate (DMA).185

The polymer has potential applications in the field of delivery

of nano-carriers after modification of its backbone using fatty acids with different lengths and

different degrees of substitution.47,122

One drawback of most synthetic comb-like polymers is the small ratio of hydrophobic

to hydrophilic entities. Increasing this ratio results in an increase of the loading capacity of

hydrophobic drugs.186

One way to increase the hydrophobicity of graft copolymers is to

synthesize amphiphilic graft copolymers with two blocks on its side chains where one of them

is hydrophobic. Again, the hydrophobic part of the side chain should also be biodegradable

when used for drug delivery.61,187

Poly(ε-caprolactone) (PCL) is semi-crystalline,

biodegradable, biocompatible with many drugs and has the ability of being fully excreted

form the body.188,189

Furthermore, amphiphilic linear block copolymers of PCL and PEO

(PCL-b-PEO) have been investigated widely for potential applications as micellar drug-


50

delivery systems.190,191

The self-assembly of PCL-b-PEO in water can vary according to the

weight fraction of PEO and to the way of preparation.192,193

One important form of these

aggregates is worm-like micelles that show strong potential for applications as nano-carriers

for hydrophobic drugs.194

Preparation of worms are usually problematic since their phase

occupies usually a narrow region of the block copolymer phase diagram.195,196

Attention has

been drawn recently to worm-like micelles caused by the fact that they have compared to

typical spherical micelles such properties as higher drug-loading capacity, longer in vivo

circulation time, and they help to shrink tumors more effectively.197,198

Nevertheless, worm-

like micelles show a relatively rapid degradation to spherical micelles as a result of hydrolytic

degradation of PCL chains initiated mainly at the hydroxyl end groups.199,200

The degradation

rate changes dramatically by end capping of the hydroxyl groups. In addition, introducing

only 10 mol% of DL-lactide within the PCL block can eliminate the rigidity of the

micelles.192

These results suggest that small modifications of the polymer composition cause

significant changes of the properties of worm-like micelles.

Generally, three strategies are applied to synthesize graft copolymers: i) “grafting

from”,36,37,201,202

ii) “grafting through”203,204

, and iii) “grafting onto”. 31,32,62,142

Amphiphilic

graft copolymer with two different side chains can be obtained either by using only one type

of grafting strategy38,205

or by a combination of two types,39,206

e.g. the combination of

“grafting from” and “grafting onto”.40,207

This chapter describes the synthesis of biodegradable graft block copolymers using

PGA as a backbone. PCL-b-PEO is attached to PGA by ring opening polymerization of ε-

caprolactone using the pendent OH groups of PGA as initiator to synthesize PGA-g-PCL with

three different lengths of the PCL chains. Then, PEO is added to PGA-g-PCL using again

CuAAC to obtain finally PGA-g-(PCL-b-PEO). For comparison, PCL-b-PEO is also

synthesized with molar masses similar to the grafted chains on the PGA backbone. The

aggregation properties of all polymers in water are extensively characterized using scattering

and microscopic techniques.


4.2.1 Materials

All chemicals were purchased from Sigma-Aldrich unless otherwise stated. Tetrahydrofuran

(THF) 99.5% was distilled from calcium hydride and stored over molecular sieve 3Å.


51

Novozym 435 was dried under vacuum at 4°C over P2O5 for two days prior to use. Tin

octoate was distilled under reduced pressure and stored over molecular sieve 4Å. Pyridine and

ε-caprolactone (99%) were dried over calcium hydride overnight, distilled under atmospheric

pressure and stored over molecular sieve (3Å). Poly(ethylene oxide) monomethyl ether =

2000 g/mol, , p-toluenesulfonyl chloride 99%, chloroform 99.9% HPLC grade, n-hexane

≥99.0%, dichloromethane 99.5%, copper bromide 99.99%, N,N,N′,N′′,N′′-

pentamethyldiethylenetriamine (PMDETA), dichloromethane ≥99.5% (DCM), N-(3-

dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N,N′-

dicyclohexylcarbodiimide (DCC), 4-(dimethylamino)pyridine (DMAP) were used as

received. Divinyl adipate (98%) was purchased from Fluorochem, U.K. and used as received.

The synthesis of azide-terminal poly(ethylene oxide) monomethylether (mPEO-N3) was

performed as described by Gao et al.31

The number-average molar mass was Mn = 2000

g/mol. Ploy(glycerol adipate) was synthesized from glycerol and DVA as described by

Kallinteri et al.122

4.2.2 Synthesis of poly(glycerol adipate)-g-poly(ε-caprolactone) PGA-g-PCL

The PGA used for this reaction had Mn= 3400 g/mol determined by SEC. PGA (1.06 g, 5.5 ×

10-3

mol with respect to OH group) was charged into a 50 mL Schlenk tube equipped with

magnetic stirrer. This was followed by addition of ε-caprolactone (15.7 mL, 0.137 mol), 0.15

mL tin octoate, and 25 mL of anhydrous THF. The solution was degassed using three freeze-

pump-thawing cycles. The resulting solution was stirred at 80°C for 20 h. Finally, the solution

was diluted with THF and precipitated in 400 mL of methanol. Precipitation in methanol was

repeated many times to remove the inevitably generated homopolymer poly(ε-caprolactone).36

The resulting polymer was dried under vacuum at room temperature. Yield=46%.

4.2.3 Synthesis of alkyne-modified poly(glycerol adipate)-g-poly(ε-caprolactone), PGA-

g- (PCL-alkyne)

PGA-g-PCL (1 g, Mn= 32000 g/mol, 0.52 mmol) and 5-hexynoic acid (0.13 mL, 1.15 mmol)

were dissolved in 25 mmol of anhydrous DCM and charged into 250 mL two neck round

bottom flask. The solution was cooled using an ice bath. Then a solution of EDCI (220 mg,

1.15 mmol) and DMAP (28 mg, 0.23 mmol) dissolved in 7 mL DCM was added dropwise.

The mixture was agitated using a magnetic stirrer and sealed using rubber a septum for 24 h at

ambient temperature. The solution was filtered to remove the precipitate. This was followed

by a concentration of the solution using rotary evaporation. The polymer solution was then


52

precipitated two times into cold diethyl ether and dried under vacuum at room temperature.

Yield=72%.

Figure 4.1 (A) Enzymatic synthesis of PGA, (B) Ring opening polymerization of ε-

caprolactone at 80°C using OH of PGA,(C)Esterification reaction to prepare

PGA-g-(PCL-alkyne), (D) CuAAC to obtain PGA-g-(PCL-b-PEO).


53

4.2.4 Synthesis of PGA-g-(PCL-b-PEO) using CuAAC

The typical procedure for the polymer synthesis can be described as the following; PGA-g-

PCL (0.550 g, Mn=32000 g/mol, 0.289 mmol), mPEO-N3 (0.618 g, Mn= 2000 g/mol, 0.301

mmol), and PMDETA (0.042 mL, 0.202 mmol) were dissolved in anhydrous DMF, and added

to 25 mL Schlenk tube. The tube was degassed by bubbling nitrogen into the solution for 20

min. This was followed by addition of CuBr (29 mg, 0.202 mmol). Further degassing was

carried out for 10 min. The solution was kept at room temperature for 48 h. The reaction was

quenched finally by addition of 10 mL THF. The polymer solution was passed through an

alumina column to remove CuBr. The resulting solution was concentrated and then dialyzed

against water for 4 days using a dialysis membrane of MWCO= 3500 g/mol. The polymer

was dried by freeze-drying. Yield=66%.

4.2.5 Synthesis of -hydroxy--alkyne end functional poly(ε-caprolactone) (Alkyne-

PCL)

The polymer was synthesized according to the procedure described by Hoogenboom et al.208

The reaction was carried out at 85 °C.

4.2.6 Synthesis of poly(ε–caprolactone)-b-poly(ethylene oxide) PCL-b-PEO

Alkyne-PCL (0.5 g, Mn=2900 g/mol, 0.172 mmol) and mPEO-N3 (0.141 g, Mn= 2000 g/mol,

0.206 mmol) were dissolved in 20 mL anhydrous DMF and added to an oven dried Schlenk

tube. The tube was sealed by rubber septum and purged with nitrogen for at least 10 min.

CuBr (15 mg, 0.1 mmol) and PMDETA (0.02 mL, 0.1 mmol) were then added. The solution

was further purged with nitrogen for 10 min. The solution was kept at room temperature for 2

days. At the end of reaction the solvent was removed under vacuum using rotary evaporator,

then 20 mL of THF was added and the solution was passed through an alumina column in

order to remove copper bromide. The solution was dialyzed against acetone for 2 days using a

dialysis membrane of MWCO=2000 g/mol. Finally, the solvent was removed and the

resulting polymer was dried in an oven at 50°C under vacuum. Yield=58%.


54

Figure 4.2 Synthesis of PCL-b-PEO. (A) Synthesis of alkyne-PCL by ring opening

polymerization in the presence of tin octoate at 100°C. (B) Coupling reaction

using CuAAC.

4.2.7 Procedures

The weight-average molar mass (Mw), number-average molar mass (Mn), and molar mass

distribution (Mw/Mn) were measured by size-exclusion chromatography (SEC) using

tetrahydrofuran (THF). The measurements were performed at room temperature using

ViscotekGPCmax VE2001 and RI detector Viscotek 3580. For calibration PS standards were

employed. 1H and

13C NMR spectra were recorded using a Varian Gemini 2000 spectrometer

operating at 400 MHz or 500 MHz for 1H NMR and 200 MHz for

13C NMR spectroscopy.

CDCl3 was used as solvent. The surface tensions γ of the aqueous polymer solution at

different concentrations were measured by the Wilhelmy plate method using an automated

DCAT tensiometer (Data Physics Instruments). The tensiometer worked automatically by

injecting predetermined volumes of micelle solution into milli Q water. The surface tension

was measured after 10 min of stirring and 3 h waiting period. Measurements were carried out

at 25° C. Dynamic light scattering (DLS) measurements were achieved using an ALV/DLS-

5000 instrument (ALV GmbH, Langen). The DLS instrument was equipped with a

goniometer for automatic measurements between scattering angles θ of 30 and 140°. The

correlation functions were analyzed by the CONTIN method, which provide information on

the distribution of decay rate (Γ). Apparent diffusion coefficients were obtained from


55

Dapp=Γ/q2 (where q=(4πn/λ) sin(θ/2), λ is the wavelength of the light, n was the refractive

index, and θ was the scattering angle). Finally, apparent hydrodynamic radii were calculated

via Stokes-Einstein equation. The solutions of polymer micelles were prepared with a

concentration of 1 g/L and directly filtered into the light scattering cells through a 0.45 µm

pore size PTFE filter. The hydrodynamic radii were determined at 12 different angles and

averaged for each concentration.

4.2.8 Micelle preparation

The typical procedure for the preparation of spherical micelles was as the following. 8 mg of

polymer was dissolved in 1.8 mL acetone. The solution was stirred for 5 h using a magnetic

stirrer. Afterwards, 3 mL of milli Q water was added to the organic solution within 3 h using a

syringe pump (KD Scientific, Holliston). The resulting solution was then transferred into a

dialysis bag (MCWO=1000 g/mol) and dialyzed against milli Q water for 24 h. On the other

hand, the micelle solutions for temperature-dependent 1H NMR experiments were prepared as

the following. 20 mg of polymer was dissolved in 0.75 mL acetone (HPLC grade) and then

stirred for 2 h. Afterwards, 2 mL of D2O was added slowly to the polymer solution under

vigorous stirring. The resulting solution was then gently stirred for 20 h at room temperature

in order to evaporate the acetone. Finally, the volume of the solution was adjusted to 1 mL by

rotary evaporator at room temperature.

4.2.9 Worm-like aggregates

The solution of worm-like aggregates was prepared by the cosolvent/evaporation method.199

Briefly, 2 mg of the graft copolymer was dissolved in 60 µl chloroform. Then the resulting

solution was added to 10 mL of milli Q water. The resulting mixture was immersed into an

ice bath and stirred for 30 min using a disperser (IKA, Type T 25 basic, Staufen, Germany) at

rotation speed of 19000 rpm. This was followed by gentle stirring for four days at 4°C or for

48 h at room temperature in order to remove chloroform. The final concentration of the

micelle solution was 0.2 mg/mL.

4.2.10 Fluorescence microscopy (FM) of worm-like aggregates

250 µL of solution with worm-like aggregates was taken in an Eppendorf tube, then 0.2 µL of

0.2 mM fluorescent dye (PKH26, Sigma) was added, the mixture was then gently mixed. 7 µL

of this solution was placed on a glass slide. The spotted solution was then covered using

round cover lip 18 mm. The worm-like aggregates were visualized using a fluorescence

microscope. Brownian motion of worm-like aggregates was visualized at 570-600 nm


56

(excitation 543 nm), using a Leica TCS SP2 DM IRE2 confocal laser scanning microscope

(CLSM) with a HCX PL APO 63x1.4 oil immersion objective (Leica Microsystems, Wetzlar,

Germany). Images were recorded from single scans or time lapse series.

4.2.11 Transmission electron microscopy (TEM), and scanning electron microscopy

(SEM)

The negatively stained samples were prepared by spreading 5 µL of the dispersion onto a Cu

grid coated with a Formvar-film (PLANO, Wetzlar). After 1 min excess liquid was blotted off

with filter paper and 5 µL of 1wt% aqueous uranyl acetate solution were placed onto the grid

and drained off after 1 min. The dried specimens were examined with an EM 900

transmission electron microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany).

Micrographs were taken with a SSCCD SM-1k-120 camera (TRS, Moorenweis, Germany).

SEM images were prepared by coating a thin layer of a micelle solution (1 mg/mL)

onto freshly cleaned silicon substrates. The water was let to evaporate at room temperature.

The substrates was immersed for 2 s into milli Q water and dried again at ambient conditions.

The sample was then coated with a 2-5 nm Pt layer by Cressington Sputter, and then

characterized by Jeol JSM-6700F field emission scanning electron microscopy


4.3.1 Synthesis and characterization of PGA-g-(PCL-b-PEO) and PCL-b-PEO

Figure 4.1 gives a summary of polymer backbones synthesis and grafting reactions carried

out. PGA is synthesized enzymatically using DVA and glycerol as monomers. The resulting

by product is vinyl alcohol which tautomerizes to acetaldehyde and thus evaporates at the

reaction temperature.

PCL is introduced to PGA by a “grafting from” strategy using ring opening

polymerization of ε-caprolactone as a monomer and tin octoate as a catalyst at different

reaction times. The hydroxyl groups on the PGA backbone initiate this reaction. Three

polymers are synthesized with different PCL molar masses. The results of the grafting

polymerizations are summarized in Table4.1.


57

Table 4.1 Mn and Mw/Mn data of PGA-g-PCL synthesized at different reaction times.

Sample Reaction

Time (h)

Mn,PCLa

(g/mol)

Mn,totalb

(g/mol)

Mw/Mnc

PGA17-g-PCL13 15 1500 28,600 1.3

PGA17-g-PCL15 20 1700 32,000 1.3

PGA17-g-PCL24 30 2700 48,800 1.5

a Mn of PCL attached to the PGA backbone calculated by

1H NMR spectroscopy.

b Mn of the

graft copolymer is obtained by 1H NMR spectroscopy.

c Obtained by SEC. All subscripts in

the sample column refer to the number of repeat monomeric units.

Actually, increasing the reaction time causes an increase of the molar mass of PCL side

chains and polydispersity. The SEC traces of PGA and PGA-g-PCL of different molar masses

are shown in Figure 4.3. The graft copolymers have higher molar masses (lower elution

volumes) than the PGA backbone. Furthermore, no trace of free PCL chains can be detected

which confirms the efficiency of the purification step.

Figure 4.3 Comparison of the SEC traces of PGA and PGA17-g-PCL13, PGA17-g-PCL15,

PGA17-g-PCL24 taken at room temperature in THF.


58

The 1HNMR spectrum of PGA17-g-PCL15 is shown in Figure 4.4. Actually, the integral ratio

between peak H` and peak D of the 1H NMR spectrum is 2 : 0.97 which indicates that the

grafting is carried out nearly quantitative on all hydroxyl groups of the PGA backbone.

Furthermore, the integral ratio between peak H` and peak E is used to calculate the length of

the grafted PCL chain. As a catalyst tin octoate is used which has FDA approval as a food

additive.209

mPEO-N3 of molar mass of 2000 g/mol is introduced to the polymer backbone by the

“grafting onto” strategy using CuAAC. This route is achieved by two steps C and D explained

in Figure 4.1. Firstly, the hydroxyl groups at the end of the PCL side chains are connected to

alkyne groups by esterification using 5-hexynoic acid. Secondly, this is followed by CuAAC

with mPEO-N3 at room temperature. Figure 4.4C reveals the complete disappearance of the

signal H` after esterification reaction with 5-hexynoic acid which essentially means that all

repeating units are linked with alkyne groups. This is followed by the coupling reaction under

mild conditions. The results of this coupling are summarized in Table 4.2.

Figure 4.4 1

H NMR spectra of (A) PGA17-g-PCL15, (B) PGA17-g-(PCL15-alkyne) and

(C)PGA17-g-(PCL15-b-PEO44) measured at room temperature in CDCl3.


59

Table 4.2 The coupling ratio, Mn, Mn,total, Mw/Mn of the polymers.

Sample

Coupling

ratioa

(mol %)

Mn,PCLb

(g/mol)

Mn,totalc

(g/mol)

Mw/Mnd

PGA17-g-(PCL15-b-

PEO44) 100 1700 65680 1.6

PGA17-g-(PCL24-b-

PEO44) 90 2700 79140 1.2

PCL16-b-PEO44 100 1800 3800 1.4

PCL25-b-PEO44 100 2900 4900 1.2

a Determined by

1H NMR,

b, c calculated using


d Obtained by SEC. All

subscripts in the sample column refer to the number of repeat monomeric units.

Typically, the ratio between peak Q and peak D in Figure 4.4 is used to determine the

coupling ratio. A comparison between the FT-IR spectra of PGA17-g-(PCL24-alkyne) and

PGA17-g-(PCL24-b-PEO44) which is shown in Figure 4.5 reveals the complete disappearance

of alkyne vibrations at 650 cm-1

and 3295 cm-1

.

Figure 4.5 FT-IR spectra of PGA17-g-(PCL24-alkyne) and PGA17-g-(PCL24-b-PEO44).Shaded

areas represent alkyne peaks.


60

The FT-IR spectrum of PGA17-g-(PCL24-b-PEO44) does not show an azide peak at 2100 cm-1

which indicates the absence of free mPEO-N3 chains from the final product.

Figure 4.6 1H NMR of (A) alkyne-PCL25 and (B) PCL25-b-PEO44 measured in CDCl3 at

500MHz.

For comparison of the properties, a linear block copolymer of PCL-b-PEO with comparable

molar masses to the respective grafted blocks at the PGA backbone is synthesized. The

synthetic route for this block copolymer is shown in Figure 4.2. CuAAC is used to couple

both blocks. The reaction is confirmed using 1H NMR spectroscopy showing the complete

disappearance of methine hydrogen resonance at δ = 2.47 ppm Figure 4.6. The comparison

between the SEC traces of alkyne-PCL25 and PCL25-b-PEO44 shows a shift towards the higher

molar mass region after coupling reaction which is shown Figure 4.7


61

Figure 4.7 SEC curves of alkyne-PCL25 and PCL25-b-PEO44 recorded using THF as eluent.

4.3.2 Dynamic Light Scattering (DLS)

The aggregation of polymers in water is characterized using DLS. Both PGA17-g-(PCL15-b-

PEO44) and PGA17-g-(PCL24-b-PEO44) are insoluble in water. However, micellar solutions of

both polymers in water are achieved by dialyzing its acetone solution against water. The DLS

results are summarized in the Table 4.3. Each radius represents the average of twelve radii

measured at different angles. Typically, the size of micelles formed by simple diblock

copolymer of PCL-b-PEO varies according to the preparation method.210

In agreement with

these results, Luo et al.211

showed that PCL23-b-PEO45 can form micelles of diameter of 25±2

nm when the micelles are prepared by dialysis from DMF solution against water. PCL16-b-

PEO44 has a shorter hydrophobic chain length hence it has a smaller micelle radius than

PCL25-b-PEO44. Both graft copolymers show two types of aggregates. The small aggregates

have similar dimensions as the micelles of the diblock copolymers and they can thus be

assigned to micelles. The intensity of the large aggregates is equal or even smaller than the

intensity of aggregates with smaller radius as shown in figure 4.8. As the intensity is

proportional to the fifth-order of radius the population of aggregates with larger radius is very

small compared to those with smaller radius.212


62

Figure 4.8 Hydrodynamic radius distribution of PGA17-g-(PCL15-b-PEO44) and PGA17-g-

(PCL24-b-PEO44) in water at a concentration of 1 g/L, scattering angle of 40°, and

temperature of 25 °C.

Table 4.3 Average of hydrodynamic radii measured in water at room temperature at

twelve different angles by DLS.

4.3.3 Micelle characterization by 1H NMR spectroscopy

A comparison between the 1H NMR spectra of PGA17-g-(PCL15-b-PEO44) obtained in D2O

and in CDCl3 is carried out. Actually, CDCl3 is considered to be a good solvent for all blocks

and the polymer backbone whereas D2O is only a good solvent for PEO.

Sample First Radius (nm) Second Radius

(nm)

PCL16-b-PEO44 9.4±0.82 ---

PCL25-b-PEO44 11.3±1.25 ---

PGA17-g-(PCL15-b-PEO44) 8.5±1 57±6

PGA17-g-(PCL24-b-PEO44) 9.5±1.2 76.3±18


63

Figure 4.9 1

H NMR spectra of PGA17-g-(PCL15-b-PEO44) obtained in D2O and in CDCl3 at

room temperature.

Figure 4.9 shows the spectra in both solvents. The comparison between both spectra reveals

that the peaks at 1.37, 1.63, 2.3, 2.75, 4.05 ppm, which belongs to the hydrophobic part of the

polymer (PGA and PCL), have a lower resolution in D2O in a comparison with the peaks in

CDCl3. Attenuation and disappearance of the splitting of signals in 1H NMR spectra indicate a

decrease of chain mobility.213–215

A reduced mobility suggests that the hydrophobic parts of

the graft copolymer form the core of micelles in water.

Figure 4.10 provides a comparison between the expanded 1H NMR spectra of PGA17-g-

(PCL15-b-PEO44) and PCL16-b-PEO44 micelles in D2O at different temperatures. The peaks in

all spectra are related to the CH2 groups of the hydrophobic PCL block and the PGA

backbone. The spectra of both polymers show a significant upfield shift of the resonance

signal with increasing temperature. The upfield shift is induced by changing of magnetic

susceptibility of the protons upon raising temperature as a result of the decreasing polarity of

water.216

Additionally, both spectra reveal that an increasing solution temperature leads also

to an increased resolution of the peaks. This is caused by an increase of the mobility of the

hydrophobic cores in the micelles of both polymers.


64

Figure 4.10 Expanded 1H NMR spectra of (A) PCL16-b-PEO44, and (B) PGA17-g-(PCL15-b-

PEO44) obtained in D2O at different temperatures.

Interestingly, the increased resolution by raising the temperature is higher for the PCL16-b-

PEO44 compared to PGA17-g-(PCL15-b-PEO44). This strongly suggests that micelles formed

by amphiphilic graft copolymers have a higher stability against temperature increase of

aqueous solutions compared to linear block copolymers of similar chemical composition of

the grafted side chains. Obviously, the polymer backbone of the graft copolymers plays a key

role in stabilizing the micelles.


65

4.3.4 Surface tension measurements

Figure 4.11 depicts the surface tension γ of aqueous solutions of PGA17-g-(PCL15-b-PEO44)

and PGA17-g-(PCL24-b-PEO44) as a function of polymer concentration at 25°. This gives the

critical micelle concentration (cmc) indicated by the intersection of the extrapolation of the

two linear regimes where the curves show a sharp change in slope. The obtained values are

0.4 µg/mL for PGA17-g-(PCL15-b-PEO44) and 1 µg/mL for PGA17-g-(PCL24-b-PEO44). Both

values are much smaller than the cmc values of PCL19-b-PEO44, PHEMA200-g-(PCL22-b-

PEO45), and PHEMA70-g-(PCL20-b-PEO45) where (PHEMA is poly(2-hydroxyethyl

methacrylate).181

Figure 4.11 Surface tension of (A) PGA17-g-(PCL15-b-PEO44) and (B) PGA17-g-(PCL24-b-

PEO44) as a function of polymer concentration at room temperature.

The cmc can be considered as indicator for micelle stability which is an important factor when

polymers are used in the field of drug delivery. According to literature,181

two factors are

proposed for the higher stability of graft copolymer micelles compared to micelles of linear

block copolymers of the same chemical structure as the grafted side chains. Firstly, the

hydrophobic interactions can be strengthened in the case of graft copolymers according to the

intermolecular interactions of PCL side chains. Secondly, PCL blocks from different graft

polymer chains are able to entangle with each other.

4.3.5 Scanning electron microscopy (SEM)

Casted and dried spherical micelles formed by PGA17-g-(PCL15-b-PEO44) in water prepared

by dialysis are shown in the SEM image of Figure 4.12. The image reveals well-dispersed

spherical micelles. They are relatively uniform in size with an average radius of 10 nm in

(A) (B)


66

agreement with DLS data. Additionally, some aggregates of micelles are observed and some

larger aggregates are also depicted in the inset of Figure 4.12 in agreement with DLS data.

Figure 4.12 SEM image of PGA17-g-(PCL15-b-PEO44) micelles. The inset shows the presence

of some aggregates larger than micelles.

4.3.6 Preparation and characterization of worm-like aggregates

Figure 4.13 shows fluorescence microscopy images of PGA17-g-(PCL24-b-PEO44) worm-like

aggregates prepared by the cosolvent/evaporation method.199

Figure 4.13 PGA17-g-(PCL24-b-PEO44) worm-like aggregates formed in water visualized by

fluorescence microscopy.


67

The hydrophobic fluorescence dye PKH26GL is loaded into the worm-like aggregates by

direct addition to the polymer solution. The fluorescence image shows the existence of worm-

like and apparently spherical aggregates. Most of the worm-like aggregates have a contour

length in the range of 2-10 µm. A vertical view of the worm-like aggregates sample is taken

by confocal laser scanning microscopy Figure 4.14. This reveals that some of the apparently

spherical aggregates are in fact also worm-like aggregates but oriented perpendicular to the

glass slide. The spherical aggregates could be attributed also to the worm aggregates that have

contour length smaller than the resolution of fluorescence microscope and thus they apear as

dots.

Figure 4.14 A vertical view of PGA17-g-(PCL24-b-PEO44) worm-like aggregates sample

taken by confocal fluorescence microscopy.

Typically, aqueous solutions of PEO containing linear block copolymers exhibit worm-like

micelle self-assembly when the weight fraction of PEO is in range of 0.42 to 0.55.192,217,218

The aqueous solution of PCL24-b-PEO44 is reported previously to form worm-like micelles.198

This block copolymer has the same chemical composition as the grafted side chain of PGA17-

g-(PCL24-b-PEO44). However, the weight fraction of PEO in the graft polymer is only 38wt%.

This suggests that this polymer will form polymersomes rather than worm-like micelle. Thus,

further investigation using TEM and SEM is necessary in order to achieve a detailed

understanding of the morphology of these worm-like aggregates.

Figures 4.15 (A) and (B) obtained by negative staining TEM show two examples of extended

worm-like aggregates. They are formed by aggregated polymersomes with an average

diameter of 25 nm. The dark circular structures are caused by an enrichment of the staining

agent in PGA rich regions of the polymersomes since PGA is an amorphous polymer and has

a lower packing density compared to PEO and PCL which are additionally able to crystalline.


68

Both TEM images show aggregates of polymersomes joined together in an elongated fashion

caused by strong shear force during the formation. This is further illustrated in Figure 4.15 (C)

The aggregates seem to have different length and different radii. Also Liu et al.219

reported

recently the occurrence of polymersomes by ternary graft copolymers and their aggregation.

Figure 4.15 (A) and (B) Typical TEM images of worm-like aggregates of PGA17-g- (PCL24-

b-PEO44) prepared by the cosolvent/evaporation method. (C) Schematic drawing

of a polymersome and the formation of worm-like aggregates.

Furthermore, tracking of Brownian motion of single worm-like aggregates with time using

CLSM is shown in Figure 4.16. Actually, the snapshots reveal a rigid behavior of the worm-

like aggregates. Thus, the worms are rigid over their entire contour. Complete or partial

rigidity of worm-like micelles of PEO-b-PCL has been explained previously as a result of

PCL crystallization within the micelle core.192

In our case, the rigidity is obviously caused by

the entanglement of PGA backbone chains as discussed below.


69

Figure 4.16 Snapshots represent the Brownian motion of a single worm-like aggregate of

PGA17-g-(PCL24-b-PEO44) in water with time using CLSM.

The overall procces to form these worm-like aggregates can be explained as follows. A

concentrated solution of the polymer in chloroform is added to water with a volume ratio of

1000 : 6 to form two immiscible layers. Both layers are stirred using a disperser at a high

revolution speed. During stirring the chloroform will begin to evaporate and the polymer

chains will thus diffuse out of the chloroform phase into the water phase where they self-

assemble as polymersomes. Actually, the temperature of the solution during the stirring

increases to 40-50°C even in the presence of an ice bath. The resulting polymersomes collide

with each other under vigorous stirring. In general, when two polymersomes which are

usually formed by block copolymers collide with each other they should fuse to form one

polymersome with larger radius. In the case of graft copolymers, the polymer backbone may

prevent the fusion process. These considerations are summarized in Figure 4.17.

The worm-like aggregates show a long time stability. Contrary to the worm-like micelles

formed by PCL-b-PEO which show a significant degradation,199

no significant changes are

noticed for the case of worm-like aggregatess formed by the graft copolymer even after weeks

when the solution is kept at 4°C. This stability can be attributed to the reduced hydrolysis of

PCL chains as a result of the absence of hydroxyl end group on both ends of the PCL block in

our graft copolymers. Additionally, the process of worm prepreation is considered as an easy,

clean and rapid process since the worms are formed spontaneously during the mixing without

the need of any surfactant. Contrary to the method suggested by He et al.220

to prepare

cylindrical micelles from PCL-b-PEO, the final product in our process contains only water as

a solvent an thus is also applicable in vivo. These worm-like aggregates have the additional

advantage, that they can be emoployed for delivering both hydrophobic and hydrophilic drugs

at the same time. Thus, the resulting worms can be considered as multi-drug carriers.

Furthermore, Discher et al.198

reported that filament particles have a significant longer

circulation time comparing to their spherical counterparts. Thus, further in vitro and in vivo


70

invistigations are necessary to achieve an understanding of these new polymers for

biomedical and pharmaceutical applications.

Figure 4.17 The structure of polymersomes formed by self-assembly of PGA17-g-(PCL24-b-

PEO44) in water and the worm-like aggregates formed by vigurous stirring during

the polymersome formation.

4.4 Conclusions

Water soluble biodegradable and biocompatible graft copolymers are prepared using

poly(glycerol adipate) (PGA) as a backbone. This backbone is synthesized by enzymatic

polymerization using divinyl adipate and glycerol to yield aliphatic linear polyester with free

pendent hydroxyl groups. PGA-g-(PCL-b-PEO) is prepared by combination of “grafting

from” and “grafting onto” methods. The later polymer has a high hydrophobic ratio which

increases drug loading capabilities. PGA-g-(PCL-b-PEO) is not water soluble but it forms

micelles by dialyzing its acetone solution against water. Spherical micelles are formed with a

hydrodynamic radius close to that formed by PCL-b-PEO with identical composition as the

graft block copolymer chains. On the other hand, micelles of PGA-g-(PCL-b-PEO) show a

higher stability at elevated temperatures compared to PCL-b-PEO of the same chemical

composition. Interestingly, PGA17-g-(PCL24-b-PEO44) can form polymersomes of an average

diameter of 25 nm. These polymersomes form worm-like aggregates caused by collision as a

result of shear forces during the preparation. The worm-like aggregates are visualized using


71

fluorescence and electron microscopy. They are shape persistent during the Brownian motion

and have potential applications as effective multi-drug carriers.

Chapter 5 Langmuir Film of Graft Copolymers

72

Chapter 5

The Behavior of Poly(ɛ-caprolactone) and Poly(ethylene oxide)-b-Poly(ɛ-

caprolactone) Grafted to a Poly(glycerol adipate) Backbone at the Air/Water

Interface

5.1 Introduction

Materials with a thickness in the nanometer range are the basis for several enabling

technogies.221

The Langmuir trough is a suitable tool to form well-organized molecular

monolayers with nanometer thickness at the air/water (A/W) interface. The Langmuir

technique can also be used to study the crystallization of liquid crystals,222–226

as well as semi-

crystalline polymers at the A/W interface.227,228

In general, the crystallization kinetics of semi-

crystalline polymers confined in thin films differs significantly when compared to bulk

samples. This is mainly attributed to interfacial interactions between thin films and substrates

which affects directly the diffusion of polymer chains toward the growth front of growing

lamellae and subsequently growth rate, melting temperature, and crystal morphology.229–241

Furthermore, using a Langmuir through coupled with light microscopy (Brewster angle and

epifluorescence microscopy) or gracing incidence X-ray measurements (GI-WAXS and GI-

SAXS) allow to study details of chain packing and crystal morphology as a function of trough

temperature, surface concentration (surface pressure) and compression rate.242

Additionally,

using ultrapure water minimizes the nucleation density for crystallization, as no surface

defects exist.243

The Langmuir trough can also be equipped with infrared reflection absorption

spectroscopy (IRRAS) to follow polymer chain orientation on a molecular level.244

Poly(ε-

caprolactone) (PCL) is a biodegradable semi-crystalline polyester with many biomedical

applications.188

The bulk crystallization of PCL has been investigated extensively since

crystallinity plays an important role for its mechanical properties and biodegradability.239,245–

250 The crystallization of PCL has also been studied in thin films and in monolayers at the

A/W interface.23,32,-33,253

In order to provide an anchor to the aqueous subphase for the

relatively hydrophobic PCL during compression at the A/W interface, it is reasonable to

attach a hydrophilic poly(ethylene oxide) block.254,255

Some research has been published

recently on the behavior of graft copolymers at the A/W interface, which contain side chains

of only one block.62,155,256

Here, we study the differences of the compression isotherms and

the morphology development at the A/W interface between PCL and PCL-b-PEO compared

to graft copolymers where PCL and PCL-b-PEO chains are attached to the backbone of


73

poly(glycerol adipate) (PGA). These graft copolymers of poly(glycerol adipate)-g-poly(ε-

caprolactone) PGA-g-PCL and poly(glycerol adipate)-g-(poly(ε-caprolactone)-b-

poly(ethylene oxide)) PGA-g-(PCL-b-PEO) have one grafted chain per PGA monomer unit.

The behavior of the graft copolymers has been studied at the A/W interface by Langmuir

trough measurements coupled with BAM. The morphology of respective Langmuir-Blodgett

films is investigated by atomic force microscopy (AFM).


5.2.1 Materials

The polymers used in this report were synthesized according to the procedure described

by Naolou et al.257

The chemical structures of the utilized graft copolymers are shown in

Figure 5.1. Table 5.1 summarizes the molar masses of the linear and graft polymers used.

Figure 5.1 Chemical structures of PGA-g-PCL and PGA-g-(PCL-b-PEO).

5.2.2 Surface Pressure Measurements

The surface pressure π as a function of mean molecular area (mmA) was measured

using a Langmuir trough (KSV, Helsinki, Finland). It was equipped with a Teflon trough and

a micro roughened platinum Wilhelmy plate. The temperature of the water subphase was

adjusted by circulating thermostated water. All experiments were carried out at 20 °C unless

otherwise stated. All samples were dissolved in chloroform prior to the placement on the

water surface. The experiments were carried out after 20 min of the placement of the polymer

solution in order to let the chloroform evaporate and the polymer chains equilibrate. The

polymer solutions were spread at different initial pressures, and different parts of the isotherm


74

were recorded in order to obtain the complete isotherms. The parts were then combined into

one plot. They overlapped within the experimental error. The compression and expansion

rates for all experiments were 750 mm2 min

-1. The static elasticity (εs) was calculated

according to Esker et al.243

by:

(

)

Table 5.1 Molar Masses and polydispersities (Mw/Mn) of the polymers

a The molar masses of PCL were calculated by


b The total molar mass was calculated using the integrals of the different peaks in

1H

NMR spectroscopy of each polymer whereas the molar mass of the backbone (PGA)

was obtained by SEC using THF as eluent.

c Polydispersities were determined by SEC using THF at room temperature and

polystyrene standards.

d The subscripts represent the degrees of polymerization.

5.2.3 Brewster Angle Microscopy (BAM)

Direct observation of the film formed by different polymers at A/W interface was carried

out using a MiniBAM instrument delivering images from a surface of around 7×5 mm with a

lateral resolution of approximately 20 µm (Nanofilm Technologie GmbH, Germany).

Sample name Mn,PCLa

(g/mol)

Mn,totalb

(g/mol)

Mw/Mnc

PCL16d

1,800 1,700 1.2

PCL25 2,800 2,800 1.3

PGA17-g-PCL15 1,700c 32,000 1.3

PGA17-g-PCL24 2,700c 48,800 1.5

PCL16-b-PEO44 1,800 3,800 1.4

PCL25-b-PEO44 2,800 4,900 1.2

PGA17-g-(PCL15-b-PEO44) 1,700 65,700 1.6

PGA17-g-(PCL24-b-PEO44) 2,700 79,100 1.2


75

5.2.4 Deposition of Langmuir–Blodgett (LB) Films

The deposition of PGA-g-PCL films formed at the A/W interface was carried out on

hydrophobically modified silicon wafer whereas the deposition of PGA-g-(PCL-b-PEO) was

achieved on hydrophilic silicon wafer. The wafer surfaces were modified and cleaned in both

cases according to the procedure described by Peetla et al.258

After treatment, silicon wafers

were then cut into small pieces of 1×1.5 cm and immersed into the water subphase of the

Langmuir trough. Polymer solutions were then spread onto the water surface. After 20 min of

equilibration, the surface was compressed using a compression rate of 750 mm2 min

-1 until the

desired transfer surface pressure was achieved. After another waiting period of 20 min, the

films were then transferred at constant surface pressure onto the silicon substrate by vertical

uptake through the films using a constant rate of 1 mm/min. Finally, the LB film was allowed

to dry in a desiccator at room temperature for 24 h.


5.3.1 The behavior of linear and grafted PCL at the A/W interface

The PGA backbone as shown in Figure 5.1 can be considered as a hydrophilic polymer

due to the pendant OH-group in every repeat unit. Nevertheless, it is not water soluble but it

swells to a large degree in water. The OH-groups can be employed for ring opening

polymerization of -caprolactone which results in PGA-g-PCL. Of course, after the

conversion of the OH-groups the polymer backbone becomes hydrophobic. Furthermore,

using ‘click’ chemistry it is possible to prepare graft block copolymers of the type PGA-g-

(PCL-b-PEO).257

Figure 5.2 shows the compression isotherms for two PCL homopolymer

samples with 16 and 25 monomer units, respectively. The inset shows the corresponding static

elasticity values, for which the mmA value was divided by the degree of polymerization to

obtain the area per monomer unit.


76

Figure 5.2 π-mmA isotherm of PCL16 (dotted line) and PCL25 (full line) measured at 20

°C. The dashed lines represent the compression-expansion hysteresis cycles of

PCL25 where (■) is first compression, (□) first expansion, (▲) second

compression and (∆) second expansion. The inset shows the static elasticity as

a function of the area per monomer unit defined as mean molecular area

divided by the degree of polymerization.

Each isotherm shows first a gentle increase of the surface pressure over an extended

mmA range upon compression. Upon further compression the slope of isotherms becomes

steeper caused by increased interactions between PCL chains. The inflection point of the first

increase in the isotherms defines also the values for the highest static elasticity εs,max at

approximately 37 Å2/ monomer unit for both PCL samples as shown in the inset of Figure 5.2.

According to the parameters of the orthorhombic unit cell of PCL (a= 0.748 ± 0.002 nm,

b= 0.498 ± 0.002 nm, c= 1.726 ± 0.003 nm) which contains two polymer chains each having

two monomer units,259,260

the surface area occupied by one monomer unit lying flat on the

water surface is approximately

(

) which is nearly the area at which

maximum elasticity εs,max occurs. The steep decrease of εs at surface areas per monomer unit

smaller than 37 Å2

is interpreted as desorption of ester groups from the water surface.243

Upon

further compression of the polymer monolayer in both isotherms an inflection point occurs


77

and the slope becomes smaller. However, a slight decrease of surface pressures with

compression occurs prior to the plateau region which is related to the fact that 3D crystals

grow faster than the 2D polymer film is compressed.261

A similar behavior has been observed

previously in the case of liquid crystals,222

amphiphilic amino acids,262

and long fatty acids

such as palmitic (C16), stearic (C18) and arachidic acid (C20).150,263–266

The reversibility of the

PCL25 isotherm is studied by measuring compression-expansion cycles. The corresponding

curves are shown in Figure 5.2. Complete reversibility over the total isotherm cannot be

studied since the trough area is limited. At the beginning of expansion a sudden drop of

surface pressure from ~12 mN/m to π ~ 8 mN/m occurs. It is followed by a plateau region

upon further expansion as a results of ‘crystal melting’, i.e. PCL monomer units leave the

crystal and reabsorb to the A/W interface.243

The surface pressure decreases again slightly

before the first compression curve is reached again, and also the second compression isotherm

shows a small shift towards smaller mmA values, which indicates that the melting process of

PCL crystals is not completely reversible and some of the polymer chains are still packed

within the crystal structure.252

The intersections of the tangents of the plateau and the steep

increase of the surface pressure at lower mmA values for the PCL16 and PCL25 isotherms are

at Ap,16 = 60 Å2 and Ap,25 = 90 Å

2, respectively. Dividing each of these numbers by the

corresponding number of monomer units per chain gives an area of ~ 3.7 Å2/monomer unit

for both polymers. This means that for both polymers the end of the plateau occurs at the

same surface area per monomer unit. This indicates that both polymer layers have the same

average thickness on the water surface at this point. Due to the semicrystalline structure it can

be expected that crystalline and amorphous regions will exist simultaneously on the water

surface. For further calculations, the cross-sectional area of a PCL segment is estimated from

the corresponding crystal dimension and is approximately

=18.6 Å

2. This indicates that in

average, including crystalline and amorphous regions, 18.6/3.7=5 monomer units are stacked

on top of each other. This height is the same for both PCL homopolymers, PCL16 and PCL25.


78

Figure 5.3 BAM images of PCL25 crystals obtained: 1) during compression at 20°C at

mmA of (a) 544 Å2 (b) 303 Å

2 and (c) 227 Å

2. 2) Relaxation at 20°C with

constant area of 214 Å2, 3)-5) Relaxation at increased temperatures (55°C,

60°C, 65°C) and 6) during cooling back to 20 °C, all at constant area of 214

Å2. Further details are given the text.

BAM images of PCL25 taken during compression at 20 °C show the appearance of

crystals (Figure 5.3 a-c). The compression stopped at mmA = 214 Å2 and the monolayer is

kept for another ~ 25 min at constant mmA. The crystals size becomes larger during this time

0.5 Compression

T= 20 °C

Relaxation

T= 20 °C

T= 55 °C

T= 60 °C

T= 65 °C

p= 38 °C

q= 27 °C

r= 20 °C

1

2

3

4

5

6


79

as shown in (Figure 5.3 c-e). The subphase temperature is then raised within 30 min to 55 °C

which is close to the melting temperature of PCL25 in bulk (Tm,PCL25=52.2 °C).267

No

significant changes of the morphology of PCL crystals can be observed (Figure 5.3 g, and h).

Only slight defects at the crystals edges can be seen in Figure 5.3 i. Then, the subphase

temperature is raised further to 60 °C for another 30 min. Figure 5.3 k reveals the beginning

of the melting process indicated by the disintegration of the crystals into small pieces.

Surprisingly, even after the apparent disappearance of the crystals two phases are observed in

the BAM images taken after 25 min of raising the subphase temperature to 60 °C Figure 5.3 l.

This might indicate the formation of a mesophase above the melting temperature of PCL. This

behavior is obviously different to the disappearance of crystals upon expansion where the

chains (ester groups) obviously reabsorb at the A/W interface. When the temperature is raised

to T=65 °C for 30 min, no significant changes were observed and the mesophase remained

(Figure 5.3 m-o). Higher temperatures cannot be employed since the movement on the water

surface becomes very fast and it is impossible to acquire BAM images. A further gradual

decrease of the subphase temperature to 20 °C produces crystals with a different morphology

(Figure 5.3 p-r) compared with the crystals formed during the first compression.

Figure 5.4 presents the compression isotherms of PGA17-g-PCL14 and PGA17-g-PCL24.

The area per PCL monomer unit was calculated according to the equation

Actually, both isotherms are similar to the isotherms of linear PCL which is also valid for

the static elasticity diagrams. The maximum of the static elasticity of εs,max is at mmA of ~ 37

Å2 and, therefore, similar to the value of linear PCL chains. However, the isotherms of both

graft copolymers do not show the slight drop in surface pressure of the isotherms prior to

reaching the plateau region. As it was mentioned above, such a drop in the isotherms indicates

that the transfer of polymer chains from the monolayer to a crystal lamella is faster than the

compression of the monolayer. Thus, the isotherms of the graft copolymers prove that the

crystallization rates in graft copolymers are slower compared to linear PCL. This seems to be

reasonable since the amorphous polymer backbone of the graft copolymers effectively hinders

the organization of the PCL graft chains into lamellae.


80

Figure 5.4 π-mmA isotherm of PGA17-g-PCL15 (full line) and PGA17-g-PCL24 (dotted

line) measured at 20°C. The dashed lines represent the compression-

expansion hysteresis cycles of PGA17-g-PCL24 where (■) is first compression,

(□) first expansion, (▲) second compression and (∆) second expansion. The

inset shows the static elasticity as a function of area per PCL monomer unit.

BAM images of the PGA17-g-PCL24 monolayer during compression indicate also another

difference compared to the crystal morphology of linear PCL (Figure 5.5 a-c). The images

show that PGA17-g-PCL24 chains form crystals with smaller size compared to linear PCL25.

Actually, similar differences between crystal sizes of linear and graft polymers in bulk have

been reported before and are interpreted as a result of increasing nucleation density of graft

polymers due to the decrease of PCL chain mobility caused by grafting.40,268

The increase of

the nucleation rate in the case of graft copolymers could also be attributed to the fact that the

grafted chains are forced to be parallel to each other on the water surface which increases the

probability of the formation of stable crystal nuclei.


81

Figure 5.5 BAM images for PGA17-g-PCL24 obtained during the hysteresis experiment

performed at 20°C. The images were obtained at surface areas of: 1-

Compression: (a) 7140 Å2, (b) 6590 Å

2, (c) 5500 Å

2. 2- Expansion: (d) 6580

Å2, (e) 9700 Å

2, (f) 16600 Å

2.

Another interesting difference between linear and grafted PCL is revealed in the

hysteresis isotherms of PGA17-g-PCL24 which are also shown in Figure 5.4. A drop of the

surface pressure from ~11 mN/m to ~1 mN/m is seen in the hysteresis isotherm of PGA17-g-

PCL24 upon expansion followed by a gradual decrease to 0 mN/m. Actually, the absence of a

plateau region (significantly above =0 mN/m) in the expansion isotherm, indicates that the

structure formed by graft copolymers is more stable than those formed by PCL resulting in a

slower re-adsorption of PCL chains at the A/W interface. Furthermore, the second

compression does not follow the first compression isotherm, but the first expansion isotherm.

This supports the above mentioned assumption that the 3D structures formed by the graft

copolymer do not melt during the expansion process. The second expansion isotherm is

similar to the first expansion. A waiting period of about 1 h between the first expansion and

second compression is also performed. However, no change of the corresponding isotherm is

noticed. The stability of crystals during expansion is also confirmed by BAM images in

(Figure 5.5 d-f).

0.5 1

2


82

5.3.2 The behavior of linear and grafted PCL-b-PEO at the A/W interface

Figure 5.6 shows the π-mmA isotherms of PCL16-b-PEO44 and PCL25-b-PEO44, PGA17-g-

(PCL15-b-PEO44), and PGA17-g-(PCL24-b-PEO44) measured at 20 °C. All isotherms show

distinct features at a surface pressure of ~ 10 mN/m. Typically, amphiphilic block copolymers

having PEO as water soluble block show a phase transition from pancake to brush

conformation at surface pressures between 9 and 13 mN/m depending on PEO block

length.62,255,258,269,270

At this surface pressure the PEO chains leave the water surface and

submerge into the water subphase. In the same surface pressure range the crystallization of

PCL blocks occurs at the A/W interface.254,255

This is further confirmed by the BAM image of

PCL25-b-PEO44 shown in the inset of Figure 5.6 A. The image reveals the presence of

filament crystals at the A/W interface. Actually, this result is consistent with the crystal

morphology reported previously for LB films formed by PCL-b-PEO.255

At higher surface pressures all four isotherms indicate a collapse of the monolayer. Only

the linear block copolymer with the smallest PCL block (PCL16-b-PEO44) shows a nearly

perfect multilayer formation, i.e. the sliding of the polymer blocks on top of each other

without any resistance (i.e. at nearly constant surface pressure).261

The other polymers show a

substantial increase of surface pressure upon compression in the roll-over region which is

most significant for PCL25-b-PEO44.40

The isotherms of PCL16-b-PEO44, PGA17-g-(PCL15-b-

PEO44), and PGA17-g-(PCL24-b-PEO44) show the plateau region at a surface pressure of ~ 30

mN/m. Such type of extended plateau at the end of isotherms has been assigned to the

collapse region which occurred either by “giant folds” or “multiple folds” or according to the

multilayer mechanism. 149–151


83

A

B

0.5

Figure 5.6 The π-mmA isotherms of (A) PCL16-b-PEO44 and PCL25-b-PEO44 linear block

copolymers. The inset shows a BAM image of PCL25-b-PEO44 taken at surface

pressure of π~ 16.5 mN/m (B) PGA17-g-(PCL15-b-PEO44), and PGA17-g-

(PCL24-b-PEO44) isotherms and the PGA17-g-(PCL24-b-PEO44) hysteresis

measurements.

The hysteresis isotherm of PGA17-g-(PCL15-b-PEO44) in Figure 5.6 B shows a short

pseudo plateau during monolayer expansion at the surface pressure of ~ 11 mN/m which can

be assigned to the melting of PCL crystals at A/W interface (i.e. the readsorption of PCL

monomer units at the A/W interface).255

Similar to linear PCL, the shift of the isotherm during

the second compression towards smaller mean molecular area values indicates that the


84

melting process does not include all PCL chains and thus some PCL chains are still packed in

the 3D structure of the PCL crystals.

Figure 5.7 (A) The π-A isotherm and hysteresis isotherm of PGA17-g-(PCL24-b-PEO44).

(B) BAM images of PGA17-g-(PCL24-b-PEO44) taken during compression-

expansion-compression cycles. For details see text.

0.5 mm

Expansion

Compression

Compression

A

B

A

B


85

BAM images are also taken during the hysteresis experiment of PGA17-g-(PCL24-b-

PEO44) in order to confirm and clarify the collapse mechanism of the monolayer (see Figure

5.7). The hysteresis experiment is carried out in the region between collapse and before

melting of PCL crystals (the pseudo plateau seen during expansion). BAM images b and c in

Figure 5.7 B reveal the presence of parallel fracture lines which appear within the polymer

monolayer. This suggests that collapse occurred according to the multilayer formation

mechanism.

Clear cracks within the polymer films appear during the expansion process which causes

the appearance of isolated condensed domains (Figure 5.7 B d-f). The BAM images reveal

also that the folds formed during the collapse process do not totally disappear during

expansion. On the other hand, the second compression shows a significant shift of the

corresponding isotherm towards smaller mmA values which confirms that the multilayers

formed during collapse do not disappear totally during expansion. Furthermore, the second

compression isotherm returns to the same point (b in Figure 5.7A) as in the first compression

indicating that no chains are desorbed from the A/W interface during collapse. It is worth

mentioning here that the monolayer is not left to relax between the hysteresis circles, and thus

the conclusions stated here are attributed to our experimental conditions.

5.3.3 Langmuir Blodgett films

Figure 5.8 depicts an AFM image and corresponding height profiles of an LB film of

PGA17-g-PCL24 taken at mmA= 7000 Å2. The image reveals the presence of disk-like crystals

of different diameter. The corresponding height profiles show that these crystals have a

homogeneous thickness of ~ 7.7 nm which matches the reported lamellar thickness formed by

linear PCL at the A/W interface and also the flat-on lamella formed after spin coating of PCL

solutions.239,252

This height corresponds to a staple of 8-9 monomer units (calculated from the

orthorhombic unit cell parameters of PCL to be c/2=1.726/2=0.863 nm per monomer unit).

Additionally, this image reveals also the presence of filament crystals on the surface of the

disk crystals with a thickness equal to multiples of approximately 7 nm. Such filaments can be

formed as distortions within the disk-like crystals during the deposition process onto silicon

substrates.


86

Figure 5.8 AFM height images of LB film of PGA17-g-PCL24 transferred at 0700 Å2 and

the corresponding height profile.

The monolayer of PGA17-g-(PCL24-b-PEO44) was transferred onto the silica substrate

at a surface pressure of 16 mN/m. The topographic AFM images reveal the presence of

branched crystals (Figure 5.9 a and b). Small platelets are joined by filament crystals. The

height profiles (Figure 5.9 c) indicate that the branched crystals have a height of ~ 7.8 nm

which is again close to the thickness of PCL crystals at A/W interface.252

Furthermore, the

height profile shows that the platelets have a thickness of ~ 15 nm which is roughly equal to

the double of the thickness of PCL crystals.

15.25 nm

7.75 nm


87

Figure 5.9 (a) and (b) AFM height image of LB films of PGA17-g-(PCL24-b-PEO44),

transferred at π = 16 mN/m. (c) Height profiles taken along the lines 1, and 2

in image (b).

5.4 Conclusions

PCL grafted onto a PGA backbone has compared to linear PCL an increased nucleation

rate, decreased crystallization rate, and produces entities with significantly smaller size at the

A/W interface. Additionally, the crystals formed by PGA17-g-PCL24 do not melt upon

expansion on the Langmuir trough at temperatures well above the melting temperature of bulk

PCL in contrast to crystals of linear PCL at the A/W interface. The AFM height image of LB

films of PGA17-g-PCL24 depicts the presence of disk-like crystals having a lamella thickness

of ~7.6 nm which matches the previously reported lamella thickness of linear PCL formed at

the A/W interface. BAM images reveal that the crystals formed by linear PCL25 form

mesophase upon raising the subphase temperature above the bulk melting point of PCL25 at

constant mean molecular area. The crystals formed after cooling the subphase to room

temperature have a different morphology than those formed at the beginning. Linear and

grafted PCL-b-PEO copolymers show one transition within their isotherms before collapse.

This transition which appears at the surface pressure of ~10 mN/m is assigned to the

dissolution of PEO chains into water subphase and to the crystallization of PCL segments at

7.8

14

2

1

a b

c


88

A/W interface. Hysteresis isotherms of PGA17-g-(PCL24-b-PEO44) show a short plateau region

during expansion due to the melting (readsorption) of PCL crystals at A/W interface.

Furthermore, the isotherms of all polymers, except PCL25-b-PEO44, show long plateau regions

at a surface pressure of ~30 mN/m. They are assigned to the collapse of the polymer

monolayer by a multilayer formation mechanism. This interpretation is further confirmed by

BAM images of PGA17-g-(PCL24-b-PEO44) within this region. Finally, the AFM image of

PGA17-g-(PCL24-b-PEO44) of LB films shows branched crystals formed by platelets and

connected by filament crystals.

Chapter 6 Summry

89

Chapter 6

Summary

The interest in the synthesis and characterization of graft copolymers has increased

recently due to their possible applications in many important fields. It has been reported that

the graft copolymers display superior properties in the field of nano-drug carriers when

compared with their linear counterparts. Biodegradability and biocompatibility properties of

polymers that are designed for biomedical applications should be considered.

The aim of the presented work was to synthesing and characterize series of novel graft

copolymers that are suitable for pharmaceutical applications. Many synthetic approaches were

applied here to achieve this target. Aliphatic polyesters with free pendent functional groups

were used always as polymer backbone to synthesize the graft copolymers.

The first part of this thesis discussed the preparation of linear aliphatic polyesters with

free pendent azide groups by enzymatic polycondensation in the presence of lipase from

Candida antarctica type B (CAL-B). The grafting reaction to the N3-functional polyester was

carried out quantitatively at room temperature using copper-catalyzed azide-alkyne

cycloaddition (CuAAC, “click” reaction) with monoalkyne-functional poly(ethylene oxide)

(alkyne-PEO, Mn = 750 g/mol). Furthermore, both enzymatic polycondensation and “click”

reaction were carried out successfully in sequential one-pot reaction. The graft copolymer was

surface-active and self-assembled in water. The graft copolymer had a critical aggregation

concentration (cac) of 3 × 10-2

µM in water determined by surface tension measurements.

Above cac, the graft copolymer formed single chains and aggregates having a hydrodynamic

radius of ∼75 nm. Furthermore, the surface activity of the polymers at the air-water interface

was studied by Langmuir trough measurements. The Langmuir isotherm of the graft polymer

showed a pseudoplateau resulting from desorption of PEO chains into the subphase upon

compression.

The second part of the thesis disscused synthesis of graft copolymers, similar to those

synthesized in the first chapter, by utilization of poly(glycerol adipate) (PGA) as polymer

backbone. PGA was synthesized by enzymatic polymerization using glycerol and either

divinyl adipate or dimethyl adipate. The PGA was linear when the enzymatic reaction was

carried out at 40°C while branching occurred at higher temperatures. The hydroxyl pendent

groups were quantitatively esterified with 5-hexynoic acid to yield PGA with alkyne

functional pendent functional groups. Afterwards, poly(ethylene oxide) monomethylether

Chapter 6 Summry

90

azide mPEO-N3 chains were quantitatively attached onto PGA using "click" reaction under

very mild condition. The linear PGA backbone was modified also by esterification with fatty

acids of different lengths yielding comb-like polymers. The melting temperature and specific

enthalpy of fusion increase with increasing degree of substitution and/or by increasing length

of the saturated fatty acids used to modify the PGA backbone. Furthermore, the comb-like

polymers have a higher thermal stability compared to the original PGA backbone. The shape

of nanoparticles prepared by an optimized interfacial deposition method depend on the type of

fatty acid used and on the degree of substitution. The nanoparticles are phase separated as a

result of the incompatibility between the polymer backbone and the teeth of the comb-like

polymers. These nanoparticles offer promising possibilities as delivery systems for lipophilic,

amphiphilic and water soluble drugs.

In the third part, poly(glycerol adipate)-graft-(poly(3-caprolactone)-block-poly(ethylene

oxide)) (PGA-g-(PCL-b-PEO)) was synthesized by ring opening polymerization of ε-

caprolactone initiated by the hydroxyl groups of PGA. This was followed by grafting of

mPEO-N3 onto the PCL by CuAAC,“click” reaction. All polymers form micelles of radii in

the range of 10 nm after dissolving in acetone and dialysis against water. Micelles formed by

PGA-g-(PCL-b-PEO) show smaller critical micelle concentration (cmc) and higher stability

against temperature increase compared to micelles formed by PCL-b-PEO with an identical

chemical composition to the grafted segments. Additionally, PGA17-g-(PCL24-b-PEO44) forms

worm-like aggregates prepared by the cosolvent/evaporation method. The resulting worm-like

aggregates were visualized by transmission electron and confocal laser scanning microscopy

and showed shape persistent behavior over their entire contour length. It is suggested that

these worm-like aggregates are formed by partially fused polymersomes under the influence

of shear flow. They have the potential for simultaneous delivery of hydrophobic and

hydrophilic drugs.

Finally, The behavior of crystallizable poly(ε-caprolactone) (PCL) and poly(ε-

caprolactone)-b-poly(ethylene oxide) (PCL-b-PEO) is studied at the air/water interface prior

and after grafting to an amorphous poly(glycerol adipate) (PGA) backbone (PGA-g-PCL,

PGA-g-(PCL-b-PEO). Langmuir isotherms are measured and the structure formation in the

monolayer films on the water surface is followed by Brewster angle microscopy (BAM) and

in Langmuir-Blodgett films after transfer to silicon substrates by atomic force microscopy

(AFM). It is observed that PGA-g-PCL forms significantly smaller crystals and has smaller

crystallization rate compared to PCL homopolymers of identical molar masses as the grafted

chains. In contrast to crystals formed by linear PCL, the crystals formed by grafted PCL in

Chapter 6 Summry

91

PGA-g-PCL do not melt (readsorb at the water surface) upon expansion on the Langmuir

trough. Additionally, raising the subphase temperature at constant surface area significantly

above the melting point of linear PCL results in the formation of a mesophase instead of the

disappearance of crystals. AFM images of Langmuir-Blodgett films reveal that PCL chains in

PGA-g-PCL and PGA-g-(PCL-b-PEO) form lamellar crystals with a disk-shape and

interconnected platelets, respectively.

Some of the chapters of this thesis are based on the following publications:

Chapter 2 based on

Naolou, T.; Busse, K.; Kressler, J. Synthesis of well-defined graft copolymers by

combination of enzymatic polycondensation and “click” chemistry.

Biomacromolecules 2010, 11, 3660–3667.

Chapter 3 based on

Naolou, T.; Weiss, V. M.; Conrad, D.; Busse, K.; Mäder, K.; Kressler, J. Fatty acid

modified poly(glycerol adipate) - polymeric analogues of glycerides, In Tailored

Polymer Architectures for Pharmaceutical and Biomedical Applications; Scholz, C.;

Kressler, J., Eds.; American Chemical Society: Washington, DC, 2013; Vol. 1135, pp.

39–52.

Chapter 4 based on

Naolou, T.; Meister, A.; Schöps, R.; Pietzsch, M.; Kressler, J. Synthesis and

characterization of graft copolymers able to form polymersomes and worm-like

aggregates. Soft Matter 2013, 9, 10364.

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105

Acknowledgments

I would like first to express my deepest gratitude and sincere appreciation to my Ph.D.

supervisor, Prof. Dr. Jörg Kressler, for his valuable guidance, motivation, suggestions and for

giving me the opportunity to work under his mentorship. He always encouraged me to

develop myself as an instructor and independent researcher which I strongly believe will help

me throughout the rest of my scientific career. It was a privilege for me to work in his

laboratory at Martin Luther University.

I am indebted to Dr. Karsten Busse for his assistance and guidance in the field of physical

chemistry of polymers. I am also grateful to Dr. Samuel Kyeremateng and Dr. Christian

Albrecht who helped me tremendously at the early phase of this work to develop my chemical

synthesis skills.

I would also like to acknowledge all current and previous members of Prof. Jörg

Kressler's group including Dr. Henning Kausche, Dr. Zofia Funke, Dr. Regina Scöps, Dr.

Sacha Reuter, Dr. Dirk Pfefferkorn, Dr. Zheng Li, Dr. Elkin Amado, Frau Claudia Hochbach

and Frau Elvira Stark. Many thanks go to Frau Susanne Tanner for the GPC measurements,

Frau Otten in Physics Department, for the IR and Raman spectroscopy and for Mr. Xiaopeng

Li for the SEM measurements.

I offer my thanks to Prof. Dr Karsten Mäder from the Pharmacy Institute and his students

Dr. Andreas Schädlich and Dipl.-Pharm. Verena Weiss for the interesting scientific

cooperation. The collaboration with Prof. Mäder group has indeed helped strengthen my

research work and made it of more value.

I would also like to thank Dr. Mohamed Farag for his encouragement and always

inspiring instructions.

I am deeply grateful to my parents who devoted and sacrificed their life to reach this point

of my study. They always raised me with a love of science and supported me in all my

scientific pursuits.

Finally, I come to address my appreciation to my wife for her love, moral support, and

kind indulgence over the years of this thesis.

106

Curriculum Vitae

Personal Details:

Name: Toufik Naolou

Date of Birth: August 1st1979

Place of Birth: Aleppo, Syria

Nationality: Syrian

Marital Status: Married

Educational Details:

06/2007 - 10/2013 Ph.D. student at the Institute of Chemistry Martin-Luther-Universität

Halle-Wittenberg, Halle (Saale), in the groups of Prof. Dr. Jörg Kreßler

(Physical Chemistry of Polymers).

09/2002 - 06/2004 Diploma of "Organic Chemistry", Aleppo University, Aleppo, Syria.

09/1998-06/2002 Bachelor of "Applied Chemistry", Aleppo University, Aleppo, Syria.

Work experience

2010-2013 Teaching Assistant, the Institute of Chemistry, Martin-Luther-

Universität, Halle-Wittenberg.

Teaching the practice of "Physikalische Chemie für Pharmazeuten"

course for Pharmacy students.

Teaching practice of "physical chemistry" for Master student

2006-2007 Teaching Assistant, Faculty of Agricultural Engineering, Al-Furate

University, Dayr az-Zawr, Syria.

Teaching "General Chemistry" and "Organic Chemistry" (Practice)

2005-2007 Senior Ink Development Chemist, "Mix Colour" to prepare inkjets,

Aleppo, Syria

2004-2005 In Charge of Fertilizer Production Unit, Al Tahhan Institution for

Manufacturing Pesticides & Fertilizers

107

Publications

1. Naolou, T.; Busse, K.; Kressler, J. Synthesis of well-defined graft copolymers by

combination of enzymatic polycondensation and “click” chemistry.


2. Schädlich, A.; Naolou, T.; Amado, E.; Schöps, R.; Kressler, J.; Mäder, K. Noninvasive

in vivo monitoring of the biofate of 195 kDa poly(vinyl alcohol) by multispectral

fluorescence imaging. Biomacromolecules 2011, 12, 3674–3683.

3. Weiss, V. M.; Naolou, T.; Hause, G.; Kuntsche, J.; Kressler, J.; Mäder, K.

Poly(glycerol adipate)-fatty acid esters as versatile nanocarriers: From nanocubes over

ellipsoids to nanospheres. J. Control. Release 2012, 158, 156–164.

4. Weiss, V. M.; Naolou, T.; Amado, E.; Busse, K.; Mäder, K.; Kressler, J. Formation of

structured polygonal nanoparticles by phase-separated comb-like polymers.

Macromol. Rapid Commun. 2012, 33, 35–40.

5. Weiss, V. M.; Naolou, T.; Groth, T.; Kressler, J.; Mäder, K. J. In vitro toxicity of

stearoyl-poly(glycerol adipate) nanoparticles. Appl. Biomater. Funct. Mater. 2012, 10,

163–169.

6. Naolou, T.; Jbeily, M.; Scholtysek, P.; Kressler, J. Synthesis and Characterization of

Stearoyl Modified Poly (Glycerol Adipate) Containing ATRP Initiator on its

Backbone. Adv. Mater. Res. 2013, 812, 1–11.

7. Pfefferkorn, D.; Pulst, M.; Naolou, T.; Busse, K.; Balko, J.; Kressler, J. Crystallization

and melting of poly(glycerol adipate)-based graft copolymers with single and double

crystallizable side chains. J. Polym. Sci. Part B Polym. Phys. 2013, DOI:

10.1002/polb.23373.

8. Naolou, T.; Meister, A.; Schöps, R.; Pietzsch, M.; Kressler, J. Synthesis and

characterization of graft copolymers able to form polymersomes and worm-like

aggregates. Soft Matter 2013, 9, 10364.

9. Jbeily, M.; Naolou, T.; Bilal, M.; Amado, E.; Kressler, J. Enzymatically synthesized

polyesters with pendant OH-groups as macroinitiators for the preparation of well-

defined graft copolymers by ATRP. Polym. Int. Accepted.

Book Chapter

1. Naolou, T.; Weiss, V. M.; Conrad, D.; Busse, K.; Mäder, K.; Kressler, J. Fatty acid

modified poly(glycerol adipate) - polymeric analogues of glycerides, In Tailored



39–52.

108

Meeting Abstracts

1. Jang, Y.; Schaedlich, A.; Schoeps, R.; Maeder, K.; Naolou, T.; Kressler, J. Poly(vinyl

alcohol) hydrogel for postsurgical adhesion prevention. 240th ACS National Meeting,

Boston, MA, United States, August 2010.

2. Naolou, T.; Busse, K.; Pietzsch, M.; Kressler, J. Modification of poly(glycerol adipate)

and its surface activity. 240th ACS National Meeting, Boston, MA, United States,

August 2010.

3. Naolou, T.; Busse, K.; Pietzsch, M.; Kressler, J. Modification of poly(glycerol adipate)

and its surface activity. Polymeric Materials (P.2010), Halle (Saale), September 2010.

4. Naolou, T.; Busse, K.; Kressler, J. Synthesis and characterization of amphiphilic graft

copolymer by combination of enzymatic polycondensation and "click" chemistry.

Polymers in Biomedicine and Electronics, Berlin, October 2010.

5. Kressler, J.; Naolou, T.; Conrad, D.; Busse, K.; Amado, E.; Weiss, V.; Mader, K. Fatty

acid modified poly(glycerol adipate)- polymeric analogues of glycerides. 243rd ACS

National Meeting, San Diego, CA, United States, March 2012.

6. Naolou, T.; Busse, K.; Kressler, J. Biocompatible graft copolymer based on

Poly(glycerol adipate). 243rd ACS National Meeting, San Diego, CA, United States,

March, 2012.

7. Naolou, T.; Kressler, J.; Schops, R. Preparation of stable worm-like aggregates using

amphiphilic biodegradable graft copolymers. 245th ACS National Meeting, New

Orleans, LA, United States, April 2013.

Oral Presentation

Naolou, T Enzymatic Synthesis of Amphiphilic Polyesters and Their Application in

Pharmacy, Polymerwerkstoffe (P2012), Halle (Saale) 2012.

109

Erklärung

Hiermit erkläre in an Eides statt, dass ich die vorliegende Arbeit selbständig und ohne

fremde Hilfe verfasst habe. Ich habe keine anderen Quellen und Hilfsmittel als die

angegebenen verwendet und anderen Werken wörtlich oder inhaltlich entnommene Stellen als

solche gekennzeichnet. Diese Arbeit habe ich an keiner anderen Hochschule vorgelegt und

mich zu keinem früheren Zeitpunkt um den Doktorgrad beworben.

Toufik Naolou

Halle (Saale), 10.12.2013

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